14.1 Glycolysis in Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway

14.1 Glycolysis

In glycolysis (from the Greek glykys, “sweet” or “sugar,” and lysis, “splitting”), a molecule of glucose is degraded in a series of enzyme-catalyzed reactions to yield two molecules of the three-carbon compound pyruvate. During the sequential reactions of glycolysis, some of the free energy released from glucose is conserved in the form of ATP and NADH. Glycolysis was the first metabolic pathway to be elucidated and is probably the best understood. From Eduard Buchner’s discovery in 1897 of fermentation in cell-free extracts of yeast until the elucidation of the whole pathway in yeast and in muscle in the 1930s, the reactions of glycolysis were a major focus of biochemical research. These discoveries showed that the reactions of life could be explained chemically, without reliance on a mystical life force. This philosophical shift led physiologist Jacques Loeb to observe in 1906, “The history of this problem is instructive, as it warns us against considering problems as beyond our reach because they have not yet found their solution.”¹

The development of methods of enzyme purification, the discovery and recognition of the importance of coenzymes such as NAD, and the discovery of the pivotal metabolic role of ATP and other phosphorylated compounds all came out of studies of glycolysis. The glycolytic enzymes of many species have long since been purified and thoroughly studied.

Glycolysis is an almost universal central pathway of glucose catabolism, the pathway with the largest flux of carbon in most cells. The glycolytic breakdown of glucose is the sole source of metabolic energy in some mammalian tissues and cell types (erythrocytes, renal medulla, brain, and sperm, for example). Some plant tissues that are modified to store starch (such as potato tubers) and some aquatic plants (watercress, for example) derive most of their energy from glycolysis; many anaerobic microorganisms are entirely dependent on glycolysis.

In the course of evolution, the chemistry of the reactions of glycolysis has been completely conserved. Genome sequencing and structural studies have shown that the glycolytic enzymes of vertebrates are closely similar in amino acid sequence and three-dimensional structure to their homologs in yeast and spinach. Although some archaea and parasitic microorganisms lack one or more of the enzymes of glycolysis, they retain the core of the pathway. The glycolytic pathway, of central importance in itself, is governed by thermodynamic principles and regulatory mechanisms that are common to all pathways of cell metabolism. It serves as a model of principles we will revisit throughout Part II of this book.

An Overview: Glycolysis Has Two Phases

Before examining each step of the pathway in some detail, we take a look at glycolysis as a whole. As all sugar derivatives in glycolysis are the d isomers, we will usually omit the d designation except when emphasizing stereochemistry. The breakdown of the six-carbon glucose into two molecules of the three-carbon pyruvate occurs in 10 steps, the first 5 of which constitute the preparatory phase (Fig. 14-2a). In these reactions, glucose is first phosphorylated at the hydroxyl group on C-6 (step ). The glucose 6-phosphate thus formed is converted to fructose 6-phosphate (step ), which is again phosphorylated, this time at C-1, to yield fructose 1,6-bisphosphate (step ). For both phosphorylations, ATP is the phosphoryl group donor.

A two-part figure shows the preparatory phase of glycolysis in part a and the payoff phase of glycolysis in part b. — FIGURE 14-2 The two phases of glycolysis. For each molecule of glucose that passes through the preparatory phase (a), two molecules of glyceraldehyde 3-phosphate are formed; both pass through the payoff phase (b). Pyruvate is the end product of the second phase of glycolysis. For each glucose molecule, two ATP are consumed in the preparatory phase and four ATP are produced in the payoff phase, giving a net yield of two ATP and two NADH per molecule of glucose converted to pyruvate. The numbered reaction steps correspond to the numbered headings in the text discussion. Keep in mind that each phosphoryl group, represented here as , has two negative charges $(- {PO}_{3}^{2 -})$ $left-parenthesis minus PO Subscript 3 Superscript 2 minus Baseline right-parenthesis$ .

FIGURE 14-2 The two phases of glycolysis. For each molecule of glucose that passes through the preparatory phase (a), two molecules of glyceraldehyde 3-phosphate are formed; both pass through the payoff phase (b). Pyruvate is the end product of the second phase of glycolysis. For each glucose molecule, two ATP are consumed in the preparatory phase and four ATP are produced in the payoff phase, giving a net yield of two ATP and two NADH per molecule of glucose converted to pyruvate. The numbered reaction steps correspond to the numbered headings in the text discussion. Keep in mind that each phosphoryl group, represented here as , has two negative charges $(- {PO}_{3}^{2 -})$ $left-parenthesis minus PO Subscript 3 Superscript 2 minus Baseline right-parenthesis$ .

Part a is labeled preparatory phase. Text below read, phosphorylation of glucose, and its conversion to glyceraldehyde 3-phosphate. It begins with glucose, shown as a six-carbon ring with the carbons numbered clockwise beginning with 1 at the right side vertex. Its has O substituted for C at the upper right vertex, C 1 at the right side vertex bonded to H above and O H below, C 2 at the lower right vertex bonded to H above and O H below, C 3 at the lower left vertex bonded to O H above and H below, C 4 at the left side vertex bonded to H above and O H below, C 5 at the upper left vertex bonded to C 6 above and H below, and C 6 bonded to 2 H and O H. Step 1 is labeled as the first priming reaction. An arrow labeled hexokinase points down from glucose and is accompanied by a curved arrow showing that an orange A T P is added and A D P is lost. This yield glucose 6-phosphate, which has a similar structure to glucose except that C 6 is bonded to 2 H and to O further bonded to an orange sphere labeled P. Step 2 shows upward and downward arrows next to phosphohexose isomerase. This yields fructose 6-phosphate, which is shown as a five-membered ring with O at the top vertex; C 2 at the right vertex bonded to C 1 above further bonded to 2 H and O H and to O H below; C 3 at the lower right vertex bonded to O H above and H below; C 4 at the lower left vertex bonded to H above and O H below; C 5 at the left side vertex bonded to C 6 above and H below; and C 6 bonded to 2 H and to O further bonded to P. Step 3 is labeled as the second priming reaction. An arrow labeled phosphofructokinase-1 points down from glucose and is accompanied by a curved arrow showing that an orange A T P is added and A D P is lost. This yields fructose 1,6- bisphosphate, shown as a similar molecule to the reactant except that C 1 is bonded to 2 H and to O further bonded to an orange sphere labeled P. The molecule is divided into two halves: a purple half on the left from the right of O at the upper right vertex to the middle of the bottom bond between C 3 and C 4 and a green half on the right. Step 4 is accompanied by text reading, cleavage of 6- carbon sugar phosphate to two 3- carbon phosphates. It shows upward and downward arrows next to aldolase. Two products are produced, glyceraldehyde 3- phosphate in purple from the left half and diacetone phosphate in green from the right half. Glyceraldehyde 3-phosphate has P bonded to O bonded to C H 2 bonded to C H bonded to O H below and to C on the right double bonded to O to the upper right and to H to the lower right. Upward and downward arrows connect this to dihydroxyacetone phosphate below, showing that they interconvert. Dihydroxyacetone phosphate has P bonded to O bonded to C H 2 bonded to C double bonded to O below bonded to C H 2 O H. Step 5 shows upward and downward arrows next to triose phosphate isomerase and extending into part b, the payoff phase. Accompanying text reads, oxidative conversion of glyceraldehyde 3-phosphate to pyruvate and the coupled formation of A T P and N A D H. Step 5 produces (2) glyceraldehyde 3-phosphate, shown as (2) P bonded to O bonded to C H 2 bonded to C H bonded to O H below and to C to the right double bonded to O to the upper right and to H to the lower right. This molecule and all of the subsequent products below are shown in purple. Step 6 is accompanied by text reading, oxidation and phosphorylation. It shows upward and downward arrows next to glyceraldehyde 3-phosphate dehydrogenase, which has a similar structure to the reactant except that the right-hand C is now double bonded to O to the upper right and bonded to O to the lower right further bonded to P. Step 7 is accompanied by text reading, first A T P- forming reaction (substrate-level phosphorylation). It shows upward and downward arrows next to phosphoglycerate kinase and a curved arrow shows that 2 A D P are added and 2 A T P are produced by the forward reaction. The product is (2) 3-phosphoglycerate, shown as (2) P bonded to O bonded to C H 2 bonded to C H bonded to O H below and to C to the right double bonded to O to the upper right and to O to the lower right. Step 8 shows upward and downward arrows next to phosphoglycerate mutase. The product is (2) 2-phosphoglycerate, shown as (2) C H 2 bonded to O H below and to C H to the right bonded to O below further bonded to P and to C to the right double bonded to O to the upper right and to O to the lower right. Step 9 shows upward and downward arrows next to enolase with an arrow branching from the downward arrow to show the loss of 2 H 2 O. The product is 2 phosphoenolpyruvate, shown as (2) C H 2 double bonded to C bonded to O below further bonded to P and to C to the right double bonded to O to the upper right and to O to the lower right. Step 10 is accompanied by text reading, second A T P- forming reaction (substrate-level phosphorylation). It shows upward and downward arrows next to pyruvate kinase, and a curved arrow shows that 2 A D P are added and 2 A T P are produced by the forward reaction. The product is (2) pyruvate, shown as (2) C H 3 bonded to C double bonded to O below and bonded to C to the right double bonded to O to the upper right and bonded to O minus to the lower right.

Fructose 1,6-bisphosphate is split to yield two different three-carbon molecules, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (step ); this is the “lysis” step that gives the pathway its name. The dihydroxyacetone phosphate is isomerized to form a second molecule of glyceraldehyde 3-phosphate (step ), ending the first phase of glycolysis. Note that two molecules of ATP are invested before the cleavage of glucose into two three-carbon pieces; there will be a good return on this investment. To summarize: in the preparatory phase of glycolysis the energy of ATP is invested, raising the free-energy content of the intermediates, and the carbon chains of all the metabolized hexoses are converted to a common product, glyceraldehyde 3-phosphate.

The energy gain comes in the payoff phase of glycolysis (Fig. 14-2b). Each molecule of glyceraldehyde 3-phosphate is oxidized and phosphorylated by inorganic phosphate (not by ATP) to form 1,3-bisphosphoglycerate (step ). Energy is then released as the two molecules of 1,3-bisphosphoglycerate are converted to two molecules of pyruvate (steps through ). Much of this energy is conserved by the coupled phosphorylation of four molecules of ADP to ATP. The net yield is two molecules of ATP per molecule of glucose used, because two molecules of ATP were invested in the preparatory phase. Energy is also conserved in the payoff phase in the formation of two molecules of the electron carrier NADH per molecule of glucose.

In the sequential reactions of glycolysis, three types of chemical transformations are particularly noteworthy: (1) degradation of the carbon skeleton of glucose to yield pyruvate; (2) phosphorylation of ADP to ATP by compounds with high phosphoryl group transfer potential, formed during glycolysis; and (3) transfer of a hydride ion to ${NAD}^{+}$ $NAD Superscript plus$ , forming NADH. The overall chemical logic of the pathway is described in Figure 14-3.

A figure shows chemical transformations in the 10 steps of glycolysis. — FIGURE 14-3 The chemical logic of the glycolytic pathway. In this simplified version of the pathway, each molecule is shown in a linear form, with carbon and hydrogen atoms not depicted, in order to highlight chemical transformations. Remember that glucose and fructose are present mostly in their cyclized forms in solution, although they are transiently present in linear form at the active sites of some of the enzymes in this pathway.

The preparatory phase, steps to , converts the six-carbon glucose into two three-carbon units, each of them phosphorylated. Oxidation of the three-carbon units is initiated in the payoff phase, steps to . To produce pyruvate, the chemical steps must occur in the order shown.

FIGURE 14-3 The chemical logic of the glycolytic pathway. In this simplified version of the pathway, each molecule is shown in a linear form, with carbon and hydrogen atoms not depicted, in order to highlight chemical transformations. Remember that glucose and fructose are present mostly in their cyclized forms in solution, although they are transiently present in linear form at the active sites of some of the enzymes in this pathway.

The preparatory phase, steps to , converts the six-carbon glucose into two three-carbon units, each of them phosphorylated. Oxidation of the three-carbon units is initiated in the payoff phase, steps to . To produce pyruvate, the chemical steps must occur in the order shown.

Glucose is shown as a horizontal line with numbered vertical lines extending from it as follows. 1: a double bond of two vertical lines extending up to O. 2: a vertical line up to O H. 3: a vertical line down to O H. 4: a vertical line up to O H. 5: a vertical line up to O H. 6: a light red box containing a vertical line up to O H. Accompanying text reads, phosphorylation of glucose ensures that pathway intermediates remain in the cell. Step 1: Phosphorylation occurs on C-6, as C-1 is a carbonyl group and cannot be phosphorylated. A downward arrow is accompanied by a curved red arrow showing the addition of A T P and loss of A D P. This yields glucose 6-phosphate, a similar molecule except that the double bond to O in position 1 is highlighted in a light red box and position 6 now has a light red box with a vertical bond to O that has a horizontal bond to a circle labeled P. Step 2: Isomerization moves the carbonyl to C-2, a prerequisite for 3 and 4. An arrow points down and to the right. This yields fructose 6-phosphate, a similar molecule except that position 1 has a vertical bond to O H above, position 2 has a double bond to O above in a light red box, and position 6 is no longer highlighted in a light red box. Step 3: C-1, now bearing a hydroxyl group, can be phosphorylated. This ensures that both products of C-C bond cleavage are phosphorylated and thus interconvertible. An arrow points down and to the left accompanied by a curved red arrow showing the addition of A T P and loss of A D P. This yields fructose 1,6-bisphosphate, which is a similar molecule except that position 1 now has a red highlighted bond to O above bonded to a circle labeled P to the left, position 2 is no longer highlighted in a light red box, and there is a wavy red line that cuts diagonally from upper left to lower right between positions 3 and 4. Step 4: The carbonyl group at C-2 facilitates C-C bond cleavage at the right location to yield two 3-carbon products by the reverse of an aldol condensation. An arrow points down to glyceraldehyde 3-phosphate, which is a horizontal line with three bonds extending upward. The left-hand and center positions are enclosed in a light red box. The left-hand position has a double bond to O above, the middle position has a bond to O H above, and the right-hand position has a bond to O with a bond to a circle labeled P to its right. Step 5: Interconversion of the two products of 4 funnels both products into a single pathway. A diagonal set of arrows showing reversible reactions point to dihydroxyacetone phosphate to the upper left, which also has three positions with bonds extending upward with the middle and right-hand positions enclosed in a light red box. The left-hand position contains O bonded to a circle labeled P to its left, the middle position has a double bond to O, and the right-hand position has a bond to O H above. Step 6: Oxidative phosphorylation of glyceraldehyde 3-phosphate, with one N A D H produced, is a prerequisite for A T P production in 7. A slightly curved downward arrow is accompanied by a curved red arrow showing the addition of N A D plus, P subscript I and loss of N A D H. This yields 1, 3-bisphosphoglycerate, which has three positions. The left-hand position has a double bond to O above and a bond to O at its lower left in a light red box and bonded to a circle labeled P to its left within the box, the middle position has a bond to O H, and the right-hand position has a bond to O with a bond to a circle labeled P to its right. Step 7: A T P production. A downward arrow is accompanied by a curved red arrow showing the addition of A D P and loss of A T P. This yields 3-phosphoglycerate, which is similar to the reactant except that O in the red box is now O minus and no longer bonded to P and the right-hand position now has a bond to O in a light red box bonded to a circle labeled P to its right within the box. Step 8: The remaining phosphoryl group moves from C-3 to C-2, setting up the final steps of the pathway. This yields 2-phosphoglycerate, which has the left-hand position double bonded to O above and bonded to O minus to the lower left with no light red box, the middle position bonded to O in a light red box and further bonded to a circle labeled P within the box, and the right-hand position bonded to O H below. Step 9: Dehydration activates the phosphoryl for transfer to A D P in 10. This yields phosphoenolpyruvate, which is similar except that the middle and right-hand positions are both in a light red box and there is a double bond between them. Step 10: A T P production. A an arrow pointing down and to the left is accompanied by a curved red arrow showing the addition of A D P and loss of A T P. This yields pyruvate, which is similar except that the middle position has a double bond to O in a light red box and there is nothing shown bonded in the right-hand position.

ATP and NADH Formation Coupled to Glycolysis

During glycolysis some of the energy of the glucose molecule is conserved in ATP, while much remains in the product, pyruvate. The overall equation for glycolysis is

Glucose + 2 {NAD}^{+} + 2 ADP + 2 P_{i} \to 2 pyruvate + 2 NADH + 2 H^{+} + 2 ATP + 2 H_{2} O

$Glucose plus 2 NAD Superscript plus Baseline plus 2 ADP plus 2 upper P Subscript i Baseline right-arrow 2 pyruvate plus 2 NADH plus 2 upper H Superscript plus Baseline plus 2 ATP plus 2 upper H Subscript 2 Baseline upper O$

(14-1)

For each molecule of glucose degraded to pyruvate, two molecules of ATP are generated from ADP and $P_{i}$ $upper P Subscript i$ , and two molecules of NADH are produced by the reduction of ${NAD}^{+}$ $NAD Superscript plus$ . The reduction of ${NAD}^{+}$ $NAD Superscript plus$ (see Fig. 13-24) proceeds by the enzymatic transfer of a hydride ion $(: H^{-})$ $left-parenthesis bold colon upper H Superscript minus Baseline right-parenthesis$ from the aldehyde group of glyceraldehyde 3-phosphate to the nicotinamide ring of ${NAD}^{+}$ $NAD Superscript plus$ , yielding the reduced coenzyme NADH. The other hydrogen atom of the substrate molecule is released to the solution as $H^{+}$ $upper H Superscript plus$ .

We can now resolve the equation of glycolysis into two processes — the conversion of glucose to pyruvate, which is exergonic:

\begin{matrix} Glucose + 2 {NAD}^{+} \to 2 pyruvate + 2 NADH + 2 H^{+} \\ Δ G_{1}^{^{'} °} = - 146 kJ/mol \end{matrix}

$StartLayout 1st Row Glucose plus 2 NAD Superscript plus Baseline right-arrow 2 pyruvate plus 2 NADH plus 2 upper H Superscript plus Baseline 2nd Row upper Delta upper G 1 Superscript Super Superscript prime Superscript degree Baseline equals negative 146 kJ slash mol EndLayout$

(14-2)

and the formation of ATP from ADP and $P_{i}$ $upper P Subscript i$ , which is endergonic:

\begin{matrix} 2 ADP + 2 P_{i} & \to 2 ATP + 2 H_{2} O \\ Δ G_{2}^{^{'} °} & = 2 (30.5 kJ/mol) = 61.0 kJ/mol \end{matrix}

$StartLayout 1st Row 1st Column 2 ADP plus 2 upper P Subscript i Baseline 2nd Column right-arrow 2 ATP plus 2 upper H Subscript 2 Baseline upper O 2nd Row 1st Column upper Delta upper G 2 Superscript Super Superscript prime Superscript degree Baseline 2nd Column equals 2 left-parenthesis 30.5 kJ slash mol right-parenthesis equals 61.0 kJ slash mol EndLayout$

(14-3)

The sum of Equations 14-2 and 14-3 gives the overall standard free-energy change of glycolysis, $Δ G_{Sum}^{^{'} °}$ $upper Delta upper G Subscript Sum Superscript prime degree$ :

\begin{matrix} Δ G_{Sum}^{^{'} °} = Δ G_{1}^{^{'} °} + Δ G_{2}^{^{'} °} & = - 146 kJ/mol + 61.0 kJ/mol \\ = - 85 kJ/mol \end{matrix}

$StartLayout 1st Row 1st Column upper Delta upper G Subscript Sum Superscript Super Superscript prime Superscript degree Baseline equals upper Delta upper G 1 Superscript Super Superscript prime Superscript degree Baseline plus upper Delta upper G 2 Superscript Super Superscript prime Superscript degree Baseline 2nd Column equals negative 146 kJ slash mol plus 61.0 kJ slash mol 2nd Row 1st Column Blank 2nd Column equals negative 85 kJ slash mol EndLayout$

Under standard conditions, and under the (nonstandard) conditions that prevail in a cell, glycolysis is an essentially irreversible process, driven to completion by a large net decrease in free energy.

Energy Remaining in Pyruvate

Glycolysis releases only a small fraction of the total available energy of the glucose molecule. The two molecules of pyruvate formed by glycolysis still contain most of the chemical potential energy of glucose, energy that can be extracted by oxidative reactions in the citric acid cycle (Chapter 16) and oxidative phosphorylation (Chapter 19) — aerobic processes. Under anaerobic conditions, pyruvate can be reduced to lactate or ethanol (Section 14.3). The oxidation of pyruvate is an important catabolic process, but pyruvate has anabolic fates as well. It can, for example, provide the carbon skeleton for the synthesis of the amino acid alanine or for the synthesis of fatty acids. We return to these anabolic reactions of pyruvate in later chapters.

Importance of Phosphorylated Intermediates

Each of the nine glycolytic intermediates between glucose and pyruvate is phosphorylated (Fig. 14-2). The phosphoryl groups have three functions.

Because the plasma membrane lacks transporters for phosphorylated sugars, the phosphorylated glycolytic intermediates cannot leave the cell. After the initial phosphorylation, no further energy is necessary to retain phosphorylated intermediates in the cell, despite the large difference in their intracellular and extracellular concentrations.
Phosphoryl groups are essential components in the enzymatic conservation of metabolic energy. Energy made available with the transfer of phosphoryl groups from compounds with phosphoanhydride bonds (such as those in ATP) is partially conserved in the formation of phosphate esters such as glucose 6-phosphate. Compounds with higher group transfer potential than ATP, which are formed in glycolysis (1,3-bisphosphoglycerate and phosphoenolpyruvate), donate phosphoryl groups to ADP to form ATP.
Binding energy resulting from the binding of phosphate groups to the active sites of enzymes lowers the activation energy and increases the specificity of the enzymatic reactions (Chapter 6). The phosphate groups of ADP, ATP, and the glycolytic intermediates form complexes with $Mg Superscript 2 plus$ $Mg Superscript 2 plus$ , and the substrate binding sites of many glycolytic enzymes are specific for these ${Mg}^{2 +}$ $Mg Superscript 2 plus$ complexes. Most glycolytic enzymes require ${Mg}^{2 +}$ $Mg Superscript 2 plus$ for activity.

The Preparatory Phase of Glycolysis Requires ATP

In the preparatory phase of glycolysis, two molecules of ATP are invested and the hexose chain is cleaved into two triose phosphates. The realization that phosphorylated hexoses were intermediates in glycolysis came slowly and serendipitously. In 1906, Arthur Harden and William Young tested their hypothesis that inhibitors of proteolytic enzymes would stabilize the glucose-fermenting enzymes in yeast extract. They added blood serum (known to contain inhibitors of proteolytic enzymes) to yeast extracts and observed the predicted stimulation of glucose metabolism. However, in a control experiment intended to show that boiling the serum destroyed the stimulatory activity, they discovered that boiled serum was just as effective at stimulating glycolysis! Careful examination and testing of the contents of the boiled serum revealed that inorganic phosphate was responsible for the stimulation. Harden and Young soon discovered that glucose added to their yeast extract was converted to a hexose bisphosphate (the “Harden-Young ester,” eventually identified as fructose 1,6-bisphosphate). This was the beginning of a long series of investigations on the role of organic esters and anhydrides of phosphate in biochemistry, which has led to our current understanding of the central role of phosphoryl group transfer in biology.

Phosphorylation of Glucose

In the first step of glycolysis (Fig. 14-2), glucose is activated for subsequent reactions by its phosphorylation at C-6 to yield glucose 6-phosphate, with ATP as the phosphoryl donor:

A chemical reaction shows the conversion of glucose to glucose 6-phosphate catalyzed by hexokinase and with Greek letter delta G prime degree symbol equals negative 16.7 k J per mole.

This reaction, which is irreversible under intracellular conditions, is catalyzed by hexokinase. Recall that kinases are enzymes that catalyze the transfer of the terminal phosphoryl group from ATP to an acceptor nucleophile (see Fig. 13-8c). Kinases are a subclass of transferases (see Table 6-3). The acceptor in the case of hexokinase is a hexose, normally glucose, although hexokinase also catalyzes the phosphorylation of other common hexoses, such as fructose and mannose, in some tissues.

Hexokinase, like many other kinases, requires ${Mg}^{2 +}$ $Mg Superscript 2 plus$ for its activity, because the true substrate of the enzyme is not ${ATP}^{4 -}$ $ATP Superscript 4 minus$ but the ${MgATP}^{2 -}$ $MgATP Superscript 2 minus$ complex (see Fig. 13-12). ${Mg}^{2 +}$ $Mg Superscript 2 plus$ shields the negative charges of the phosphoryl groups in ATP, making the terminal phosphorus atom an easier target for nucleophilic attack by an $— OH$ $em-dash OH$ of glucose. Hexokinase undergoes a profound change in shape, an induced fit, when it binds glucose; two domains of the protein move about 8 Å closer to each other when ATP binds (see Fig. 6-30). This movement brings bound ATP closer to a molecule of glucose also bound to the enzyme and blocks the access of water (from the solvent), which might otherwise enter the active site and attack (hydrolyze) the phosphoanhydride bonds of ATP. Like the other nine enzymes of glycolysis, hexokinase is a soluble, cytosolic protein.

Hexokinase is present in nearly all organisms. The human genome encodes four different hexokinases (I to IV), all of which catalyze the same reaction, but differ in kinetics, regulation, and location. Two or more enzymes that catalyze the same reaction but are encoded by different genes are called isozymes (see Box 14-3). One of the isozymes present in hepatocytes, hexokinase IV (also called glucokinase), differs from other forms of hexokinase in kinetic and regulatory properties, with important physiological consequences that are described in Section 14.5.

Conversion of Glucose 6-Phosphate to Fructose 6-Phosphate

The enzyme phosphohexose isomerase (phosphoglucose isomerase) catalyzes the reversible isomerization of glucose 6-phosphate, an aldose, to fructose 6-phosphate, a ketose:

The mechanism for this reaction involves an enediol intermediate (Fig. 14-4). The reaction proceeds readily in either direction, as might be expected from the relatively small change in standard free energy.

A figure shows the mechanism of the phosphohexose isomerase reaction in four steps. — MECHANISM FIGURE 14-4 The phosphohexose isomerase reaction. The ring opening and closing reactions (steps and ) are catalyzed by an active-site His residue, by mechanisms omitted here for simplicity. The proton (light red) initially at C-2 is made more easily abstractable by electron withdrawal by the adjacent carbonyl and nearby hydroxyl groups. After its transfer from C-2 to the active-site Glu residue (a weak acid), the proton is freely exchanged with the surrounding solution; that is, the proton abstracted from C-2 in step is not necessarily the same one that is added to C-1 in step .

MECHANISM FIGURE 14-4 The phosphohexose isomerase reaction. The ring opening and closing reactions (steps and ) are catalyzed by an active-site His residue, by mechanisms omitted here for simplicity. The proton (light red) initially at C-2 is made more easily abstractable by electron withdrawal by the adjacent carbonyl and nearby hydroxyl groups. After its transfer from C-2 to the active-site Glu residue (a weak acid), the proton is freely exchanged with the surrounding solution; that is, the proton abstracted from C-2 in step is not necessarily the same one that is added to C-1 in step .

Glucose 6-phosphate is a six-membered ring with O at the top right vertex in the sixth position of the ring, the carbons numbered, and C 1 bonded to H above and O H below, C 2 and C 4 bonded to H above and O H below, C 3 bonded to O H above and H below, C 5 bonded to C 6 above and H below, and C 6 bonded to 2 H and to O P O subscript 3 superscript 2 minus. A dashed line is shown across the upper right bond, which is between O and C 1. Upward- and downward-pointing arrows indicate a reversible reaction. Step 1: Binding and opening of the ring. A curved border representing the active site at the right side of phosphohexose isomerase is shown to the left of a vertical molecule with numbered carbons. C 1 is bonded to H to the upper left and double bonded to O to the upper right, C 2 is bonded to H in a light red box on the left and to O on the right bonded to H in a light blue box, C 3 is bonded to O H on the left and H on the right, C 4 is bonded to H on the left and O H on the right, C 5 is bonded to H on the left and O H on the right, and C 6 is bonded to H 2 and O P O subscript 3 superscript 2 minus. B embedded in the active site of phosphohexose isomerase has a red pair of electrons with a curved red arrow pointing to the H in a light red box bonded to C 2. Additional curved red arrows point from the bond between the H in the light red box and the bond between C 1 and C 2 and from the double bond between C 1 and O to the same O. Right- and left-pointing arrows indicate a reversible reaction. H minus in a yellow box is added to the right-pointing arrow. Step 2: Proton abstraction by active-site G l u (B with a red pair of electrons) leads to italicized cis end italics-enediol formation. This yields the italicized cis end italics-enediol intermediate, which is a similar molecule in which B in the active site of phosphohexose isomerase is bonded to H in a light red box, O bonded to the upper right of C 1 is bonded to H in a yellow box, and there is a double bond between C 1 and C 2. Red curved arrows point from the double bond between C 1 and C 2 to the H in the light red box bonded to B and from the bond between O bonded to H in a light blue box and the bond between the same O and C 2. An arrow points right with an arrow curving away to show the loss of H minus in a light blue box. Step 3: General acid catalysis by the same G l u facilitates formation of fructose 6-phosphate. This yields a similar molecule in which C 1 is bonded to H in a light red box to the left, to H above, and to O to the right further bonded to H in a yellow box and C 2 is double bonded to O to its right. B has a red pair of electrons again. Upward- and downward-pointing arrows indicate a reversible reaction. Step 4: Dissociation and closing of the ring. This yields fructose 6-phosphate, which is a five-membered ring with O at the top vertex, the carbons numbered, and C 2 at the right vertex of the ring bonded to C 1 above further bonded to 2 H and O H and to O H below, C 3 bonded to O H above and H below, C 4 bonded to H above and O H below, C 5 bonded to C 6 above and H below, and C 6 bonded to 2 H and to O P O subscript 3 superscript 2 minus. There is a dashed line across the upper right bond between O and C 2.

Phosphorylation of Fructose 6-Phosphate to Fructose 1,6-Bisphosphate

In the second of the two priming reactions of glycolysis, phosphofructokinase-1 (PFK-1) catalyzes the transfer of a phosphoryl group from ATP to fructose 6-phosphate to yield fructose 1,6-bisphosphate:

A chemical reaction shows the conversion of fructose 6-phosphate to fructose 1,6-bisphosphate catalyzed by phosphofructokinase-1 (P F K-1) and with Greek letter delta G prime degree symbol equals negative 14.2 k J per mole.

Fructose 6-phosphate, which is a five-membered ring with O at the top vertex, the carbons numbered, and C 2 at the right vertex of the ring bonded to C 1 above further bonded to 2 H and O H in a light red box and to O H below, C 3 bonded to O H above and H below, C 4 bonded to H above and O H below, C 5 bonded to C 6 above and H below, and C 6 bonded to 2 H and to O P O subscript 3 superscript 2 minus. An arrow points right above phosphofructokinase-1 (P F K-1) beneath a curved arrow showing the addition of A T P and loss of A D P with M g 2 plus shown at its inflection point against the right-pointing arrow. This yields fructose 1,6-bisphosphate, which is similar except that C 1 is bonded to H 2 and to O in a light red box further bonded to P O subscript 3 superscript 2 minus within the box.

Key convention

Compounds that contain two phosphate or phosphoryl groups attached at different positions in the molecule are named bisphosphates (or bisphospho compounds); for example, fructose 1,6-bisphosphate and 1,3-bisphosphoglycerate. Compounds with two phosphates linked together as a pyrophosphoryl group are named diphosphates; for example, adenosine diphosphate (ADP). Similar rules apply for the naming of trisphosphates (such as inositol 1,4,5-trisphosphate; see p. 425) and triphosphates (such as adenosine triphosphate, ATP).

The enzyme that forms fructose 1,6-bisphosphate is called PFK-1 to distinguish it from a second enzyme (PFK-2) that catalyzes the formation of fructose 2,6-bisphosphate from fructose 6-phosphate in a separate pathway (the roles of PFK-2 and fructose 2,6-bisphosphate are discussed in Section 14.5). The PFK-1 reaction is essentially irreversible under cellular conditions, and it is the first “committed” step in the glycolytic pathway; glucose 6-phosphate and fructose 6-phosphate have other possible fates, but fructose 1,6-bisphosphate is targeted for glycolysis.

Phosphofructokinase-1 is subject to complex allosteric regulation; its activity is increased whenever the cell’s ATP supply is depleted or when the ATP breakdown products, ADP and AMP (particularly the latter), accumulate. The enzyme is inhibited whenever the cell has ample ATP and is well supplied by other fuels such as fatty acids. In some organisms, fructose 2,6-bisphosphate (not to be confused with the PFK-1 reaction product, fructose 1,6-bisphosphate) is a potent allosteric activator of PFK-1. Ribulose 5-phosphate, an intermediate in the pentose phosphate pathway discussed in Section 14.6, also activates phosphofructokinase indirectly. The multiple layers of regulation of this step in glycolysis are discussed in greater detail in Section 14.5.

Some bacteria and protists and perhaps all plants have a different phosphofructokinase (PP-PFK-1) that uses pyrophosphate $left-parenthesis PP Subscript i Baseline right-parenthesis$ $left-parenthesis PP Subscript i Baseline right-parenthesis$ , not ATP, as the phosphoryl group donor in the synthesis of fructose 1,6-bisphosphate:

\begin{matrix} Fructose 6 -phosphate + {PP}_{i} \overset{{Mg}^{2 +}}{\to} fructose 1,6 -bisphosphate + P_{i} \\ Δ G^{'} ° = - 2.9 kJ/mol \end{matrix}

$StartLayout 1st Row Fructose 6 hyphen phosphate plus PP Subscript i Baseline right-arrow Overscript Mg Superscript 2 plus Baseline Endscripts fructose 1 comma 6 hyphen bisphosphate plus upper P Subscript i Baseline 2nd Row upper Delta upper G Superscript prime Baseline degree equals negative 2.9 kJ slash mol EndLayout$

We will discuss this enzyme in Chapter 20.

Cleavage of Fructose 1,6-Bisphosphate

The enzyme fructose 1,6-bisphosphate aldolase, often called simply aldolase, catalyzes a reverse aldol condensation (Fig. 14-5; see Fig. 13-4). Fructose 1,6-bisphosphate is cleaved to yield two different triose phosphates, namely, glyceraldehyde 3-phosphate (an aldose) and dihydroxyacetone phosphate (a ketose):

A chemical reaction shows the conversion of fructose 1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate catalyzed by aldolase and with Greek letter delta G prime degree symbol equals negative 23.8 k J per mole.

Fructose 1,6-bisphosphate is a five-membered ring with O at the top vertex, the carbons numbered, and C 2 at the right vertex of the ring bonded to C 1 above further bonded to 2 H and O P O subscript 3 superscript 2 minus and to O H below, C 3 bonded to O H above and H below, C 4 bonded to H above and O H below, C 5 bonded to C 6 above and H below, and C 6 bonded to 2 H and to O P O subscript 3 superscript 2 minus. Right-and left-pointing arrows are shown above aldolase, indicating a reversible reaction. This yields two products shown as vertical chains with their carbons labeled with numbers in parentheses. Dihydroxyacetone phosphate has the C labeled (1) bonded to 2 H and O P O subscript 3 superscript 2 minus, C (2) double bonded to O, and C (3) bonded to 2 H and O H. Glyceraldehyde 3-phosphate has C (4) double bonded to O to the upper left and bonded to H to the upper right, C (5) bonded to H and O H, and C (6) bonded to 2 H and O P O subscript 3 superscript 2 minus.

A figure shows the class Roman numeral 1 aldolase reaction that converts fructose 1,6-bisphosphate into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate in 5 steps. — MECHANISM FIGURE 14-5 The class I aldolase reaction. Note that cleavage between C-3 and C-4 depends on the presence of the carbonyl group at C-2, which is converted to an imine on the enzyme. A and B represent amino acid residues that serve as general acid (A) or base (B).

Fructose 1,6-bisphosphate is a five-membered ring with O at the top vertex and C 2 at the right vertex of the ring bonded to C 1 above further bonded to 2 H and O P O subscript 3 superscript 2 minus and to O H below, C 3 bonded to O H above and H below, C 4 bonded to H above and O H below, C 5 bonded to C 6 above and H below, and C 6 bonded to 2 H and to O P O subscript 3 superscript 2 minus. A dashed line is shown across the upper right bond between O and C 2. Upward- and downward-pointing arrows indicate a reversible reaction, and accompanying text reads, binding and opening of the ring. This yields a vertical molecule inside of a rectangular cutout in the top of a larger structure labeled aldolase. The vertical molecule has its carbons numbered. C 1 is bonded to 2 H and O P O subscript 3 superscript 2 minus, C 2 is double bonded to O on the right, C 3 is bonded to O H on the left and H on the right, C 4 is bonded to H on the left and O bonded to H on the right, C 5 is bonded to H on the left and O H on the right, and C 6 is bonded to 2 H and O P O subscript 3 superscript 2 minus. At the upper left border of the cutout, N has a red pair of electrons and is bonded to H above and below and to L y s to the left. Below it, the wall of the cutout invaginates slightly and then continues to run vertically. The region below contains B at its boundary with a red pair of electrons and further bonded to the left. A similar B with a red pair of electrons is in the middle of the right boundary of aldolase and further bonded to the right. Just above it, H in the cutout region has a bond across the boundary of aldolase to A that is further bonded to the right. Curved red arrows point from the red electrons on N at the upper left to C 2 and from the double bond between C 2 and O to the H bonded to A in the upper right boundary of aldolase. Right- and left-pointing arrows are shown to the right, indicating a reversible reaction. The right-pointing arrow is joined by a line indicating the addition of blue highlighted H plus. Step 1: Active-site L y s attacks substrate carbonyl, leading to formation of tetrahedral intermediate. This yields a product shown in brackets. The linear molecule is similar to the reactant except that C 2 is bonded to N minus to the left that is bonded to H above and below and further bonded to the left and is bonded to blue highlighted O on the right further connected by a blue bond to blue highlighted H. On the right boundary of aldolase, A has a red pair of electrons. Red curved arrows point from the red electrons on B on the lower left boundary of aldolase to the H above it bonded to N minus above, from the bond between this H and N to the bond between this N and C 2, and from the bond between C 2 and blue highlighted O to blue highlighted H plus above. Right- and left-pointing arrows are shown to the right, indicating a reversible reaction. The right-pointing arrow has an arrow branching away to show the loss of blue highlighted H 2 O. Step 2: Rearrangement leads to formation of protonated Schiff base on enzyme; electron delocalization facilitates subsequent steps. The product is labeled protonated Schiff base. N embedded in the upper left boundary of aldolase has a positive charge, is bonded to H above, and is bonded to L y s to the left. B below it is bonded to H to the right and further bonded to the left. A embedded in the upper right boundary of aldolase has a red pair of electrons and is further bonded to the right and B below it has a red pair of electrons and is further bonded to the right. The vertical molecule has a similar structure except that C 2 is double bonded to N to its left. Curved red arrows point from the red pair of electrons on B at the lower right boundary of aldolase to H bonded to O bonded to C 4 and from the bond between this H and O and the bond between the same O and C 4, from the bond between C 4 and C 3 to the bond between C 3 and C 2, and from the double bond between C 2 and N plus to N plus to N plus. Upward- and downward-pointing arrows indicate a reversible reaction. An arrow branching from the downward arrow is labeled first product released and points to glyceraldehyde 3-phosphate, which is shown in a light red box as a three-carbon vertical chain with C 1 bonded to H to the upper left and double bonded to O to the upper right, C 2 bonded to H on the left and to O H on the right, and C 3 bonded to 2 H and to O P O subscript 3 superscript 2 minus. Step 3: C to C bond cleavage (reverse of aldol condensation) leads to release of first product. The product below is a three-carbon vertical chain with C 1 bonded to 2 H and to O P O subscript 3 superscript 2 minus, C 2 is bonded to N in the upper left side of the boundary of the aldolase that has a red pair of electrons and is bonded to H above and L y s to the left, C 2 double bonded to C 3, and C 3 bonded to O H to the lower left and H to the lower right. B on the lower left side of the aldolase boundary is bonded to H in the opening to its right and further bonded to the left, B at the lower right side of the aldolase boundary is bonded to H in the opening to its left and further bonded to the right, and A at the upper right side of the aldolase boundary has a red pair of electrons and is further bonded to the right. Curved red arrows point from the red pair of electrons on N to the bond between N and C 2 and from the double bond between C 2 and C 3 to the bond between B at the lower left side of the cutout and H. Right- and left-pointing arrows are shown to the left, indicating a reversible reaction. Step 4: Isomerization. This yields a similar molecule and cutout boundary except that C 2 is double bonded to N plus to the left that is bonded to H above and L y s to the left, there is a single bond between C 2 and C 3, C 3 is bonded to O H to the left and H below and to the right, and B at the lower left boundary of the cutout has a red pair of electrons. Right- and left-pointing arrows are shown to the left, indicating a reversible reaction. An arrow curves from H 2 O, to the right-pointing arrow, to indicate that the second product is released. This is dihydroxyacetone, which has a three-carbon vertical chain with C 1 bonded to 2 H and O P O subscript 3 superscript 2 minus, C 2 double bonded to O, and C 3 bonded to 2 H and O H. Step 5: Schiff base is hydrolyzed in reveres of Schiff base formation. This yields the cutout with no vertical chain inside with N at the upper left boundary with a red pair of electrons, bonded to H above and below, and bonded to L y s to the left, B at the lower left boundary with a red pair of electrons and further bonded to the left, B at the lower right boundary with a red pair of electrons and further bonded to the right, and A at the upper right boundary bonded to H to the left and further bonded to the right.

There are two classes of aldolases. Class I aldolases, found in animals and plants, use the mechanism shown in Figure 14-5. Class II enzymes, in fungi and bacteria, do not form the Schiff base intermediate. Instead, a zinc ion at the active site is coordinated with the carbonyl oxygen at C-2; the ${Zn}^{2 +}$ $Zn Superscript 2 plus$ polarizes the carbonyl group and stabilizes the enolate intermediate created in the $C — C$ $upper C em-dash upper C$ bond cleavage step (see Fig. 6-23).

Although the aldolase reaction has a strongly positive standard free-energy change in the direction of fructose 1,6-bisphosphate cleavage, at the lower concentrations of reactants present in cells the actual free-energy change is small and the aldolase reaction is readily reversible. We shall see later that aldolase acts in the reverse direction during the process of gluconeogenesis.

Interconversion of the Triose Phosphates

Only one of the two triose phosphates formed by aldolase, glyceraldehyde 3-phosphate, can be directly degraded in the subsequent steps of glycolysis. The other product, dihydroxyacetone phosphate, is immediately and reversibly converted to glyceraldehyde 3-phosphate by the fifth enzyme of the glycolytic sequence, triose phosphate isomerase:

A chemical reaction shows the conversion of dihydroxyacetone phosphate to glyceraldehyde 3-phosphate catalyzed by triose phosphate isomerase and with Greek letter delta G prime degree symbol equals 7.5 k J per mole.

The reaction mechanism is similar to the reaction promoted by phosphohexose isomerase in step of glycolysis (Fig. 14-4). After the triose phosphate isomerase reaction, the carbon atoms derived from C-1, C-2, and C-3 of the starting glucose are chemically indistinguishable from C-6, C-5, and C-4, respectively (Fig. 14-6); both “halves” of glucose have yielded glyceraldehyde 3-phosphate.

A two-part figure shows the fate of glucose carbons in the formation of glyceraldehyde 3-phosphate with part a showing how the carbons are distributed when fructose 1,6-bisphosphate is converted to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate and part b summarizing the source of each carbon in glyceraldehyde 3-phosphate. — FIGURE 14-6 Fate of the glucose carbons in the formation of glyceraldehyde 3-phosphate. (a) The origin of the carbons in the two three-carbon products of the aldolase and triose phosphate isomerase reactions. The end product of the two reactions is glyceraldehyde 3-phosphate (two molecules). (b) Each carbon of glyceraldehyde 3-phosphate is derived from either of two specific carbons of glucose. Note that the numbering of the carbon atoms of glyceraldehyde 3-phosphate differs from that of the glucose from which it is derived. In glyceraldehyde 3-phosphate, the most complex functional group (the carbonyl) is specified as C-1. This numbering change is important for interpreting experiments with glucose in which a single carbon is labeled with a radioisotope. (See Problems 5 and 22 at the end of this chapter.)

Part a shows fructose 1,6-phosphate as a vertical six-carbon chain with numbered carbons and with the top half in a light red box and the bottom half in a light blue box. In the light red box, C 1 is bonded to 2 H and to O further bonded to a circle labeled P, C 2 is double bonded to O to the right, and C 3 is bonded to O H to the left and to H to the right. In the light blue box, C 4 is bonded to H on the left and O H on the right, C 5 is bonded to H on the left and O H on the right, and C 6 is bonded to 2 H and to O further bonded to a circle labeled P. A double-headed arrow labeled aldolase points downward and splits to point to a light red box on the left and a light blue box on the right. The light red box is labeled dihydroxyacetone and is a vertical three-carbon chain. To the left, a column is labeled, derived from glucose carbon. The top carbon is derived from glucose carbon 1 and bonded to O further bonded to P in a circle. The middle carbon is derived from glucose carbon 2. The bottom carbon is derived from glucose carbon 3 and bonded to 2 H and O H. The light blue box is labeled glyceraldehyde 3-phosphate and is also a vertical three-carbon chain. To the right, a column is labeled, derived from glucose carbon. The top carbon is derived from glucose carbon 4, bonded to H on the left, and double bonded to O on the right. The middle carbon is derived from glucose carbon 5, bonded to H on the left, and bonded to O H on the right. The bottom carbon is derived from glucose carbon 6 and is bonded to 2 H and O further bonded to a circle labeled P. A curved arrow points from dihydroxyacetone phosphate to glyceraldehyde 3-phosphate and a similar arrow points in the reverse direction above text reading, triose phosphate isomerase. Part b shows a vertical three-carbon chain labeled glyceraldehyde 3-phosphate. A column to the left is labeled, derived from glucose carbons. The carbons are numbered. C 1 is derived from glucose carbon 4 or 3, bonded to H on the left, and double bonded to O on the right. C 2 is derived from glucose carbon 5 or 2, bonded to H on the left, and bonded to O H on the right. C 3 is derived from glucose carbon 6 or 1 and is bonded to 2 H and O further bonded to a circle labeled P. An arrow points down to text reading, subsequent reactions of glycolysis.

This reaction completes the preparatory phase of glycolysis. The hexose molecule has been phosphorylated at C-1 and C-6 and then cleaved to form two molecules of glyceraldehyde 3-phosphate.

The Payoff Phase of Glycolysis Yields ATP and NADH

The payoff phase of glycolysis (Fig. 14-2b) includes the energy-conserving phosphorylation steps in which some of the chemical energy of the glucose molecule is conserved in the form of ATP and NADH. Remember that one molecule of glucose yields two molecules of glyceraldehyde 3-phosphate, and both halves of the glucose molecule follow the same pathway in the second phase of glycolysis. The conversion of two molecules of glyceraldehyde 3-phosphate to two molecules of pyruvate is accompanied by the formation of four molecules of ATP from ADP. However, the net yield of ATP per molecule of glucose degraded is only two, because two ATP were invested in the preparatory phase of glycolysis to phosphorylate the two ends of the hexose molecule.

Oxidation of Glyceraldehyde 3-Phosphate to 1,3-Bisphosphoglycerate

The first step in the payoff phase is the oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate, catalyzed by glyceraldehyde 3-phosphate dehydrogenase:

A chemical reaction shows the addition of glyceraldehyde 3-phosphate and inorganic phosphate to produce 1,3-bisphosphoglycerate catalyzed by glyceraldehyde 3-phosphate dehydrogenase and with Greek letter delta G prime degree symbol equals 6.3 k J per mole.

Glyceraldehyde 3-phosphate is a three-carbon vertical chain. It has the top C double bonded to O to the upper left and bonded to H to the upper right, the middle C bonded to H and O H, and the bottom C bonded to 2 H and O P O subscript 3 superscript 2 minus. Inorganic phosphate is added, which is shown almost entirely within a light red box. It has P double bonded to O above, bonded to O minus to the right and below, and bonded to O to the left within the box that is bonded to H outside of the box. Right- and left-pointing arrows are shown above triose phosphate isomerase, indicating a reversible reaction. A curved arrow that joins these arrows shows that N A D plus is added and N A D H and H plus leave. This yields 1,3-bisphosphoglycerate, which has a similar structure to glyceraldehyde 3-phosphate except that the top C is double bonded to O to the left and bonded to O in a light red box to the upper right further bonded within the box to P double bonded to P above and, bonded to O minus to the right and below.

This is the first of the two energy-conserving reactions of glycolysis that eventually lead to the formation of ATP. The aldehyde group of glyceraldehyde 3-phosphate is oxidized, not to a free carboxyl group but to a carboxylic acid anhydride with phosphoric acid. This type of anhydride, called an acyl phosphate, has a very high standard free energy of hydrolysis ( $Δ G^{'} ° = - 49.3 kJ/mol$ $upper Delta upper G Superscript prime Baseline degree equals negative 49.3 kJ slash mol$ ; see Table 13-6). Much of the free energy of oxidation of the aldehyde group of glyceraldehyde 3-phosphate is conserved by formation of the acyl phosphate group at C-1 of 1,3-bisphosphoglycerate.

Glyceraldehyde 3-phosphate is covalently bound to the dehydrogenase during the reaction (Fig. 14-7). The aldehyde group of glyceraldehyde 3-phosphate reacts with the $— SH$ $em-dash SH$ group of an essential Cys residue in the active site, in a reaction analogous to the formation of a hemiacetal (see Fig. 7-5), in this case producing a thiohemiacetal. Reaction of the essential Cys residue with a heavy metal such as ${Hg}^{2 +}$ $Hg Superscript 2 plus$ irreversibly inhibits the enzyme.

A figure shows the mechanism of the glyceraldehyde 3-phosphate dehydrogenase reaction in five steps. — MECHANISM FIGURE 14-7 The glyceraldehyde 3-phosphate dehydrogenase reaction.

An irregular concave border that is horizontal at the lower right represents a piece of the right side of glyceraldehyde 3-phosphate dehydrogenase. N A D plus is shown in the opening to its upper right and S minus is shown in its horizontal boundary at the lower right. S minus is bonded to C y s below, within the enzyme. An arrow points to the right and a line shows that glyceraldehyde 3-phosphate is added. Step 1: Formation of enzyme-substrate complex. The active-site C y s has a reduced p K subscript a (5.5 instead of 8) when N A D plus is bound, and is in the more reactive, thiolate form. This yields a similar illustration in which a linear vertical molecule is above S minus. The structure of the molecule is as follows. The top carbon is bonded to 2 H and to O P O subscript 3 superscript 2 minus plus, the middle carbon is bonded to H to the left and to O H to the right, and the bottom carbon is bonded to H to the lower left and double bonded to O to the lower right. Curved red arrows point from the negative charge on S to the bottom carbon and from the double bond between the bottom carbon and O to the same O. An arrow points downward. Step 2: A covalent thiohemiacetal linkage forms between the substrate and the bonded S minus group of the C y s residue. This yields a similar molecule in which the bottom carbon is bonded to H to the left, bonded to O minus to the right, and bonded to S below in the border of the enzyme and further bonded to C y s within the enzyme. Curved red arrows point from the negative charge on O to its bond with the bottom carbon and from the bond between the bottom carbon and H to its left and to N A D plus near the upper left side of the boundary of the enzyme. An arrow points downward. Step 3: The enzyme-substrate intermediate is oxidized by the N A D plus bound to the active site. This yields a similar illustration in which N A D plus has become N A D H and the bottom carbon is double bonded to O to the right and bonded to S below further bonded to C y s. An arrow points to the left with an arrow showing the loss of N A D H and two lines to its left showing the addition of N A D plus and red highlighted P subscript i. Step 4: The N A D H product leaves the active site and is replaced by another molecule of N A D plus. This yields a similar illustration in which N A D H has become N A D plus again. A red highlighted molecule of phosphate is shown as P double bonded to O above, bonded to O H to the right, and bonded to O minus below and to the left. Red curved arrows point from the left-hand O minus of the phosphate to the bottom carbon and from the bond between the bottom carbon and S to the S. An arrow points upward with an arrow branching away to show the loss of 1,3-bisphosphoglycerate. This molecule has its top carbon bonded to 2 H and to O P O subscript 3 superscript 2 minus, its middle carbon bonded to H on the left and O H on the right, and its bottom carbon double bonded to O to the right and bonded to red highlighted O below further bonded to red highlighted P O subscript 3 superscript 2 minus. Step 5: The covalent thioester linkage between the substrate and enzyme undergoes phosphorolysis (attack by P subscript i), releasing the second product, 1,3-bisphosphoglycerate. This yields the starting illustration of glyceraldehyde 3-phosphate dehydrogenase.

The amount of ${NAD}^{+}$ $NAD Superscript plus$ in a cell $(\leq 10^{- 5} M)$ $left-parenthesis less-than-or-equal-to 10 Superscript negative 5 Baseline upper M right-parenthesis$ is far smaller than the amount of glucose metabolized in a few minutes. Glycolysis would soon come to a halt if the NADH formed in this step of glycolysis were not continuously reoxidized and recycled. We return to a discussion of this recycling of ${NAD}^{+}$ $NAD Superscript plus$ later in the chapter.

Phosphoryl Transfer from 1,3-Bisphosphoglycerate to ADP

The enzyme phosphoglycerate kinase transfers the high-energy phosphoryl group from the carboxyl group of 1,3-bisphosphoglycerate to ADP, forming ATP and 3-phosphoglycerate:

A chemical reaction shows the addition of 1,3 bisphosphoglycerate and A D P to produce 3-phosphoglycerate and A T P catalyzed by phosphoglycerate kinase and with Greek letter delta G prime degree symbol equals negative 18.8 k J per mole.

1,3-bisphosphoglycerate is a three-carbon vertical chain. It has the top C double bonded to O to the upper left and bonded to O further bonded to P within a light red box that is double bonded to O above and bonded to O minus to the right and below, all within the box. Its middle C is bonded to H and O H and its bottom C is bonded to 2 H and O P O subscript 3 superscript 2 minus. Upward- and downward-pointing arrows are shown with M g 2 plus to the left and phosphoglycerate kinase to the right, indicating a reversible reaction. This yields 3-phosphoglycerate and A T P. 3-phosphoglycerate has a similar structure to 1,3-bisphosphoglycerate except that the top carbon is double bonded to O to the left and bonded to O minus to the right. A T P has a chain with O at the bottom bonded to a rectangle labeled adenosine to its right and to a circle labeled P above bonded to another circle labeled P bonded to P in a light red box further double bonded to O to the right and bonded to O minus above and to the left, all within the box.

Notice that phosphoglycerate kinase is named for the reverse reaction, in which it transfers a phosphoryl group from ATP to 3-phosphoglycerate. Like all enzymes, it catalyzes the reaction in both directions. This enzyme acts in the direction suggested by its name during gluconeogenesis (see Fig. 14-16) and during photosynthetic ${CO}_{2}$ $CO Subscript 2$ assimilation (see Fig. 20-26). In glycolysis, the reaction it catalyzes proceeds as shown above, in the direction of ATP synthesis.

Steps and of glycolysis together constitute an energy-coupling process in which 1,3-bisphosphoglycerate is the common intermediate; it is formed in the first reaction (which would be endergonic in isolation), and its acyl phosphate group is transferred to ADP in the second reaction (which is strongly exergonic). The sum of these two reactions is

\begin{matrix} Glyceraldehyde 3 -phosphate + ADP + P_{i} + {NAD}^{+} ⇌ \\ 3 -phosphoglycerate + ATP + NADH + H^{+} Δ G^{'} ° = - 12.2 kJ/mol \end{matrix}

$StartLayout 1st Row Glyceraldehyde 3 hyphen phosphate plus ADP plus upper P Subscript i Baseline plus NAD Superscript plus Baseline right harpoon over left harpoon 2nd Row 3 hyphen phosphoglycerate plus ATP plus NADH plus upper H Superscript plus Baseline upper Delta upper G Superscript prime Baseline degree equals negative 12.2 kJ slash mol EndLayout$

Thus the overall reaction is exergonic.

Recall from Chapter 13 that the actual free-energy change, $Δ G$ $upper Delta upper G$ , is determined by the standard free-energy change, $Δ G^{'} °$ $upper Delta upper G prime degree$ , and the mass-action ratio, Q, which is the ratio [products]/[reactants] (see Eqn 13-4). For step ,

\begin{matrix} Δ G & = Δ G^{'} ° + R T ln Q \\ = Δ G^{'} ° + R T ln \frac{[1, 3 -bisphosphoglycerate] [NADH]}{[glyceraldehyde 3 -phosphate] [P_{i}] [{NAD}^{+}]} \end{matrix}

$StartLayout 1st Row 1st Column upper Delta upper G 2nd Column equals upper Delta upper G Superscript prime Baseline degree plus upper R upper T ln upper Q 2nd Row 1st Column Blank 2nd Column equals upper Delta upper G Superscript prime Baseline degree plus upper R upper T ln StartFraction left-bracket 1 comma 3 hyphen bisphosphoglycerate right-bracket left-bracket NADH right-bracket Over left-bracket glyceraldehyde 3 hyphen phosphate right-bracket left-bracket upper P Subscript i Baseline right-bracket left-bracket NAD Superscript plus Baseline right-bracket EndFraction EndLayout$

Notice that $[H^{+}]$ $left-bracket upper H Superscript plus Baseline right-bracket$ is not included in Q. In biochemical calculations, $[H^{+}]$ $left-bracket upper H Superscript plus Baseline right-bracket$ is assumed to be a constant $left-parenthesis 10 Superscript negative 7 Baseline upper M right-parenthesis$ $left-parenthesis 10 Superscript negative 7 Baseline upper M right-parenthesis$ , and this constant is included in the definition of $Δ G^{'} °$ $upper Delta upper G prime degree$ (p. 468).

When the mass-action ratio is less than 1.0, its natural logarithm has a negative sign. In the cytosol, where these reactions are taking place, the ratio $[NADH] / [{NAD}^{+}]$ $left-bracket NADH right-bracket slash left-bracket NAD Superscript plus Baseline right-bracket$ is a small fraction, contributing to a low Q. Step , by consuming the product of step (1,3-bisphosphoglycerate), keeps [1,3-bisphosphoglycerate] relatively low in the steady state and thereby keeps Q for the overall energy-coupling process small. When Q is small, the contribution of ln Q can make $Δ G$ $upper Delta upper G$ strongly negative. This is simply another way of showing how the two reactions, steps and , are coupled through a common intermediate.

The outcome of these coupled reactions, both reversible under cellular conditions, is that the energy released on oxidation of an aldehyde to a carboxylate group is conserved by the coupled formation of ATP from ADP and $P_{i}$ $upper P Subscript i$ . The formation of ATP by phosphoryl group transfer from a substrate such as 1,3-bisphosphoglycerate is referred to as a substrate-level phosphorylation, to distinguish this mechanism from respiration-linked phosphorylation. Substrate-level phosphorylations involve soluble enzymes and chemical intermediates (1,3-bisphosphoglycerate in this case). Respiration-linked phosphorylations, on the other hand, involve membrane-bound enzymes and transmembrane gradients of protons (Chapter 19).

Conversion of 3-Phosphoglycerate to 2-Phosphoglycerate

The enzyme phosphoglycerate mutase catalyzes a reversible shift of the phosphoryl group between C-2 and C-3 of glycerate; ${Mg}^{2 +}$ $Mg Superscript 2 plus$ is essential for this reaction:

The reaction occurs in two steps (Fig. 14-8). A phosphoryl group initially attached to a His residue of the mutase is transferred to the hydroxyl group at C-2 of 3-phosphoglycerate, forming 2,3-bisphosphoglycerate (2,3-BPG). The phosphoryl group at C-3 of 2,3-BPG is then transferred to the same His residue, producing 2-phosphoglycerate and regenerating the phosphorylated enzyme. Phosphoglycerate mutase is initially phosphorylated by phosphoryl transfer from 2,3-BPG, which is required in small quantities to initiate the catalytic cycle and is continuously regenerated by that cycle.

A figure shows the phosphoglycerate mutase reaction in two steps. — MECHANISM FIGURE 14-8 The phosphoglycerate mutase reaction.

A structure is rectangular on the left with rounded sides and narrows to a point on the right. This is labeled phosphoglycerate mutase. Inside, there is a three-carbon vertical chain labeled 3-phosphoglycerate that has the following structure: the top carbon forms C O O minus, the middle carbon is bonded to H to the left and O to the right further bonded to H, and the bottom carbon is bonded to 2 H and to O on the right further bonded to blue highlighted P O subscript 3 superscript 2 minus. A five-membered ring is shown to the upper right within the structure. It is positioned at an angle so that the top vertex is slightly to the left and the vertex that would normally be at the upper left side is instead at the left side. It has N plus substituted for C at its left side bonded to red highlighted P O subscript 3 superscript 2 minus, a double bond between this and C at the lower left side, a single bond to N substituted for C at its lower right side and bonded to H, a single bond between this N and C at the upper right side, a double bond between this C and C at the upper left, and C at the upper left bonded to the left, to a chain with one bend that connects to H i s outside of the enzyme. A second five-membered ring below has N with a red pair of electrons substituted for C at its top vertex, N substituted for C and bonded to H at its lower left vertex, double bonds at its upper left and lower right sides, and a chain with one bend that extends from its lower right vertex to H i s. Curved red arrows point from the bond between O bonded to the middle C of the vertical chain and H to the red highlighted P of the top five-membered ring, from the bond between this P and N plus to N plus, and from the red pair of electrons on N in the bottom ring to H bonded to O bonded to the middle carbon of the vertical chain. Upward- and downward-pointing arrows indicate a reversible reaction. Step 1: Phosphoryl transfer occurs between an active-site H i s and C-2 (O H) of the substrate. A second active-site H i s acts as general base catalyst. This yields a similar molecule labeled 2,3-bisphosphoglycerate (2,3-B P G) in which the middle carbon is bonded to O bonded to red highlighted P O subscript 3 superscript 2 minus. The top five-membered ring now has a red pair of electrons on the N that had formerly been bonded to red highlighted P O subscript 3 superscript 2 minus. The bottom ring now has N at the top vertex bonded to H and with a positive charge. Curved red arrows point from the red pair of electrons on N in the upper ring to blue highlighted P bonded to O bonded to the bottom carbon of the chain and from the bond between this P and O to H bonded to N plus of the bottom ring. Upward- and downward-pointing arrows indicate a reversible reaction. Step 2: Phosphoryl transfer from C-3 of the substrate to the first active-site H i s. The second active-site H i s acts as a general acid catalyst. This yields a similar illustration in which the bottom carbon of the linear chain is bonded to 2 H and to O on the right further bonded to H, the top five-membered ring has N at the left side carrying a positive charge and bonded to blue highlighted P O subscript 3 superscript 2 minus, and N at the top vertex of the bottom ring with no H, charge, or pair of electrons.

Dehydration of 2-Phosphoglycerate to Phosphoenolpyruvate

In the second glycolytic reaction that generates a compound with high phosphoryl group transfer potential (the first was step ), enolase promotes reversible removal of a molecule of water from 2-phosphoglycerate to yield phosphoenolpyruvate (PEP):

A chemical reaction shows the conversion of 2-phosphoglycerate to phosphoenolpyruvate catalyzed by enolase and with Greek letter delta G prime degree symbol equals 7.5 k J per mole.

2-phosphoglycerate is a three-carbon vertical chain with the top C double bonded to O to the upper left and bonded to O minus to the upper right, the middle C bonded to H in a light red box to the left and O P O subscript 3 superscript 2 minus to the right, and the bottom C bonded to 2 H and to O H in a light red box to the left. Right- and left-pointing arrows are shown with enolase below, indicating a reversible reaction. An arrow curving away from the right-pointing arrow shows the loss of H 2 O in a light red box. This yields phosphoenolpyruvate, which has a similar structure except that the middle C is bonded to O P O subscript 2 superscript 2 minus to the right, the middle and bottom C atoms are connected by a double bond highlighted in a light red box, and the bottom C is bonded to 2 H.

The mechanism of the enolase reaction involves an enolic intermediate stabilized by ${Mg}^{2 +}$ $Mg Superscript 2 plus$ (see Fig. 6-31). The reaction converts a compound with a relatively low phosphoryl group transfer potential $(Δ G^{'} °$ $left-parenthesis upper Delta upper G prime degree$ for hydrolysis of 2-phosphoglycerate is $- 17.6 kJ/mol)$ $negative 17.6 kJ slash mol right-parenthesis$ to one with high phosphoryl group transfer potential $(Δ G^{'} °$ $left-parenthesis upper Delta upper G prime degree$ for PEP hydrolysis is $- 61.9 kJ/mol)$ $negative 61.9 kJ slash mol right-parenthesis$ (see Fig. 13-13).

Transfer of the Phosphoryl Group from Phosphoenolpyruvate to ADP

The last step in glycolysis is the transfer of the phosphoryl group from phosphoenolpyruvate to ADP, catalyzed by pyruvate kinase, which requires $K^{+}$ $upper K Superscript plus$ and either ${Mg}^{2 +}$ $Mg Superscript 2 plus$ or $Mn Superscript 2 plus$ $Mn Superscript 2 plus$ :

A chemical reaction shows the addition of phosphoenolpyruvate and A D P catalyzed by pyruvate kinase to yield pyruvate and A T P with Greek letter delta G prime degree symbol equals negative 31.4 k J per mole.

Phosphoenolpyruvate is a three-carbon vertical chain with the top C double bonded to O to the upper left and bonded to O minus to the upper right; the middle C bonded to O further bonded to P in a colored box double bonded to O above and bonded to O minus to the right and below, all in the box; a double bond between the middle and bottom C atoms; and the bottom C bonded to 2 H. A D P is shown as O bonded to a rectangle labeled adenosine to the right and to a circle labeled P above further bonded to another circle labeled P. A downward arrow has M g 2 plus, K plus on the left and pyruvate kinase on the right. This yields pyruvate and A D P. Pyruvate is a three-carbon chain with the top C double bonded to O to the upper left and bonded to O minus to the upper right, the middle C double bonded to O to the right, and the bottom C bonded to 3 H. A T P is similar to A D P, except that the top circle labeled P is bonded to P in a colored box further double bonded to O to the right and bonded to O minus above and to the left, all within the box.

In this substrate-level phosphorylation, the product pyruvate first appears in its enol form, then tautomerizes nonenzymatically to its keto form, which predominates at pH 7:

A figure shows the conversion between the enol and keto forms of pyruvate through tautomerization.

The overall reaction has a large, negative standard free-energy change, due in large part to the spontaneous conversion of the enol form of pyruvate to the keto form (see Fig. 13-13). About half of the energy released by PEP hydrolysis $(Δ G^{'} ° = - 61.9 kJ/mol)$ $left-parenthesis upper Delta upper G Superscript prime Baseline degree equals negative 61.9 kJ slash mol right-parenthesis$ is conserved in the formation of the phosphoanhydride bond of ATP $(Δ G^{'} ° = - 30.5 kJ/mol)$ $left-parenthesis upper Delta upper G Superscript prime Baseline degree equals negative 30.5 kJ slash mol right-parenthesis$ , and the rest $( - 31.4 kJ/mol)$ $left-parenthesis zero width space negative 31.4 kJ slash mol right-parenthesis$ constitutes a large driving force pushing the reaction toward ATP synthesis. We discuss the regulation of pyruvate kinase in Section 14.5.

The Overall Balance Sheet Shows a Net Gain of Two ATP and Two NADH Per Glucose

We can now construct a balance sheet for glycolysis to account for (1) the fate of the carbon skeleton of glucose, (2) the input of $P_{i}$ $upper P Subscript i$ and ADP and output of ATP, and (3) the pathway of electrons in the oxidation-reduction reactions. The left side of the following equation shows all the inputs of ATP, ${NAD}^{+}$ $NAD Superscript plus$ , ADP, and $P_{i}$ $upper P Subscript i$ (consult Fig. 14-2), and the right side shows all the outputs (keep in mind that each molecule of glucose yields two molecules of pyruvate):

\begin{matrix} Glucose + 2 ATP + 2 {NAD}^{+} + 4 ADP + 2 P_{i} \to \\ 2 pyruvate + 2 ADP + 2 NADH + 2 H^{+} + 4 ATP + 2 H_{2} O \end{matrix}

$StartLayout 1st Row Glucose plus 2 ATP plus 2 NAD Superscript plus Baseline plus 4 ADP plus 2 upper P Subscript i Baseline right-arrow 2nd Row 2 pyruvate plus 2 ADP plus 2 NADH plus 2 upper H Superscript plus Baseline plus 4 ATP plus 2 upper H Subscript 2 Baseline upper O EndLayout$

Canceling out common terms on both sides of the equation gives the overall equation for glycolysis:

\begin{matrix} Glucose + 2 {NAD}^{+} + 2 ADP + 2 P_{i} \to \\ 2 pyruvate + 2 NADH + 2 H^{+} + 2 ATP + 2 H_{2} O \end{matrix}

$StartLayout 1st Row Glucose plus 2 NAD Superscript plus Baseline plus 2 ADP plus 2 upper P Subscript i Baseline right-arrow 2nd Row 2 pyruvate plus 2 NADH plus 2 upper H Superscript plus Baseline plus 2 ATP plus 2 upper H Subscript 2 Baseline upper O EndLayout$

In the overall glycolytic process, one molecule of glucose is converted to two molecules of pyruvate (the pathway of carbon). Two molecules of ADP and two of $P_{i}$ $upper P Subscript i$ are converted to two molecules of ATP (the pathway of phosphoryl groups). Four electrons, as two hydride ions, are transferred from two molecules of glyceraldehyde 3-phosphate to two of ${NAD}^{+}$ $NAD Superscript plus$ (the pathway of electrons).

SUMMARY 14.1 Glycolysis

Glycolysis is a near-universal pathway by which a glucose molecule is oxidized, in two phases, to two molecules of pyruvate, with energy conserved as ATP and NADH. Ten cytosolic enzymes act sequentially in glycolysis. The overall reaction converts glucose to two molecules of pyruvate, and energy is conserved in the synthesis of two molecules of ATP and two molecules of NADH.
In the preparatory phase of glycolysis, two molecules of ATP are invested to activate glucose to fructose 1,6-bisphosphate. The bond between C-3 and C-4 is then broken to yield two molecules of triose phosphate.
In the payoff phase, each of the two molecules of glyceraldehyde 3-phosphate derived from glucose undergoes oxidation at C-1; some of the energy of this oxidation reaction is conserved in the form of one NADH and two ATP per triose phosphate oxidized.
Subtracting the two ATP spent in the preparatory phase, the net equation for the overall process is
$\begin{matrix} Glucose + 2 {NAD}^{+} + 2 ADP + 2 P_{i} \to \\ 2 pyruvate + 2 NADH + {2 H}^{+} + 2 ATP + {2 H}_{2} O \end{matrix}$ $StartLayout 1st Row Glucose plus 2 NAD Superscript plus Baseline plus 2 ADP plus 2 upper P Subscript i Baseline right-arrow 2nd Row 2 pyruvate plus 2 NADH plus 2 upper H Superscript plus Baseline plus 2 ATP plus 2 upper H Subscript 2 Baseline upper O EndLayout$