Garrett R.H., Grisham C.M. - Biochemistry (1999)(2nd ed.)(en)

Glucose

P

NAD+

NADH

ADP

ATP

ADP

ATP

ATP	first
ADP	priming
	reaction
	Glucose-6-phosphate
	(G6P)

Fructose-6-phosphate

(F6P)

ATP

second ADP priming reaction

Fructose-1,6-bisphosphate

(FBP)

	Dihydroxyacetone phosphate
	(DHAP)
Glyceraldehyde-3-phosphate	Glyceraldehyde-3-phosphate
(G3P)	(G3P)
1,3-bisphosphoglycerate	1,3-bisphosphoglycerate
(BPG)	(BPG)
first	first
ATP-forming	ATP-forming
reaction	reaction
3-phosphoglycerate (3PG)	3-phosphoglycerate (3PG)
2-phosphoglycerate (2PG)	2-phosphoglycerate (2PG)
H2O	H2O
Phosphoenolpyruvate (PEP)	Phosphoenolpyruvate (PEP)
second	second
ATP-forming	ATP-forming
reaction	reaction

2 Pyruvate

P

NAD+

NADH

ADP

ATP

ADP

ATP

Phase 1

Phosphorylation of glucose and conversion to 2 molecules of glyceraldehyde- 3-phosphate;

2 ATP are used to prime these reactions.

Phase 2

Conversion of glyceraldehyde- 3-phosphate to pyruvate and coupled formation of 4 molecules of ATP.

	2NAD+	2CoASH
	2NADH	2 CO2
	Aerobic			Anaerobic	2 NADH	Anaerobic	2 NADH
	conditions 2 AcetylCoA			conditions		conditions
					2 NAD+		2 NAD+
					2 Lactate		2 Ethanol
		TCA					+
		cycle					2 CO2
			4	CO2
	Animals and plants			+	Anaerobic glycolysis	Alcoholic fermentation
			4	H2O
	in aerobic conditions						in yeast
				in contracting muscle

FIGURE 19.1 ● The glycolytic pathway.

611

FIGURE 19.2

612 Chapter 19 ● Glycolysis

		Glucose
	ATP		first
	ADP		priming
			reaction
		Glucose-6-phosphate
		(G6P)
		Fructose-6-phosphate
	ATP	(F6P)
			second
	ADP		priming
			reaction
		Fructose-1,6-bisphosphate
		(FBP)
			Dihydroxyacetone phosphate
			(DHAP)
	Glyceraldehyde-3-phosphate		Glyceraldehyde-3-phosphate
P		(G3P)	(G3P)	P
NAD+				NAD+
NADH				NADH
	1,3-bisphosphoglycerate		1,3-bisphosphoglycerate
		(BPG)	(BPG)
ADP		first	first	ADP
		ATP-forming	ATP-forming
ATP		reaction	reaction	ATP
	3-phosphoglycerate (3PG)		3-phosphoglycerate (3PG)
	2-phosphoglycerate (2PG)		2-phosphoglycerate (2PG)
	H2O		H2O
	Phosphoenolpyruvate (PEP)		Phosphoenolpyruvate (PEP)
ADP		second	second	ADP
		ATP-forming	ATP-forming
ATP		reaction	reaction	ATP

2 Pyruvate

● In the first phase of glycolysis, five reactions convert a molecule of glucose to two molecules of glyceraldehyde-3-phos- phate.

In the first five steps of glycolysis, one six-carbon molecule of glucose is split into two

3-carbon compounds.

2 molecules of ATP are required to prime these reactions.

6CH2OH

H		5			O			H
		H								D-Glucose
4									1
		OH			H
HO								OH
				3	2
		H			OH

ATP

Hexokinase

Mg2+ glucokinase

ADP

H

1C

O

H

C

OH

D-Glucose-6-phosphate (G6P)

HO

C

H

H

C

OH

H

C

OH

6CH2

O

PO23–

Phosphoglucoisomerase

1

CH2OH

C

O

HO

C

H

H

C

OH

D-Fructose-6-phosphate (F6P)

H

C

OH

ATP

6CH2

O

PO23–

Mg2+

Phosphofructokinase

ADP

1CH2

O

PO23–

C

O

HO

C

H

D-Fructose-1,6-bisphosphate (FBP)

Aldol cleavage

H

C

OH

H

C

OH

6CH2

O

PO23–

Fructose

bisphosphate

aldolase

1 CH2

Dihydroxyacetone

O

PO23–

H

D-Glyceraldehyde-

2 C

O

4 C

O

phosphate (DHAP)

3-phosphate (G3P)

3

CH2OH

H

5 C

OH

6

Triose phosphate

CH2

O

PO23–

isomerase

Glycolysis couples these two reactions:

Glucose 2ADP 2Pi 88n 2 lactate 2ATP 2H 2H2O (19.3)G° 183.6 61 122.6 kJ/mol

Thus, under standard-state conditions, (61/183.6) 100%, or 33%, of the free energy released is preserved in the form of ATP in these reactions. However, as we discussed in Chapter 3, the various solution conditions, such as pH, concentration, ionic strength, and presence of metal ions, can substantially alter the free energy change for such reactions. Under actual cellular conditions, the free energy change for the synthesis of ATP (Equation 19.2) is much larger, and approximately 50% of the available free energy is converted into ATP. Clearly, then, more than enough free energy is available in the conversion of glucose into lactate to drive the synthesis of two molecules of ATP.

19.3 ● The First Phase of Glycolysis

One way to synthesize ATP using the metabolic free energy contained in the glucose molecule would be to convert glucose into one (or more) of the highenergy phosphates in Table 3.3 that have standard-state free energies of hydrolysis more negative than that of ATP. Those molecules in Table 3.3 that can be synthesized easily from glucose are phosphoenolpyruvate, 1,3-bisphos- phoglycerate, and acetyl phosphate. In fact, in the first stage of glycolysis, glucose is converted into two molecules of glyceraldehyde-3-phosphate. Energy released from this high-energy molecule in the second phase of glycolysis is then used to synthesize ATP.

Reaction 1: Phosphorylation of Glucose by Hexokinase or Glucokinase—The First Priming Reaction

The initial reaction of the glycolysis pathway involves phosphorylation of glucose at carbon atom 6 by either hexokinase or glucokinase. The formation of such a phosphoester is thermodynamically unfavorable and requires energy input to operate in the forward direction (Chapter 3). The energy comes from ATP, a requirement that at first seems counterproductive. Glycolysis is designed to make ATP, not consume it. However, the hexokinase, glucokinase reaction (Figure 19.2) is one of two priming reactions in the cycle. Just as old-fashioned, hand-operated water pumps (Figure 19.3) have to be primed with a small amount of water to deliver more water to the thirsty pumper, the glycolysis pathway requires two priming ATP molecules to start the sequence of reactions and delivers four molecules of ATP in the end.

The complete reaction for the first step in glycolysis is

-D-Glucose ATP4 88n -D-glucose-6-phosphate2 ADP3 H (19.4)G° 16.7 kJ/mol

The hydrolysis of ATP makes 30.5 kJ/mol available in this reaction, and the phosphorylation of glucose “costs” 13.8 kJ/mol (see Table 19.1). Thus, the reaction liberates 16.7 kJ/mol under standard-state conditions (1M concentrations), and the equilibrium of the reaction lies far to the right (K eq 850 at 25°C; see Table 19.1).

Under cellular conditions, this first reaction of glycolysis is even more favorable than at standard state. As pointed out in Chapter 3, the free energy change for any reaction depends on the concentrations of reactants and products.

19.3 ● The First Phase of Glycolysis

613

FIGURE 19.3 ● Just as a water pump must be “primed” with water to get more water out, the glycolytic pathway is primed with ATP in steps 1 and 3 in order to achieve net production of ATP in the second phase of the pathway.

(Michelle Sassi/The Stock Market)

614 Chapter 19 ● Glycolysis

Table 19.1

Reactions and Thermodynamics of Glycolysis

Reaction Enzyme

-D-Glucose ATP4 34 glucose-6-phosphate2 ADP3 H

Glucose-6-phosphate2 34 fructose-6-phosphate2

Fructose-6-phosphate2 ATP4 34 fructose-1,6-bisphosphate4 ADP3 H

Fructose-1,6-bisphosphate4 34 dihydroxyacetone-P2 glyceraldehyde-3-P2

Dihydroxyacetone-P2 34 glyceraldehyde-3-P2

Glyceraldehyde-3-P2 Pi2 NAD 34 1,3-bisphosphoglycerate4 NADH H

1,3-Bisphosphoglycerate4 ADP3 34 3-P-glycerate3 ATP4

3-Phosphoglycerate3 34 2-phosphoglycerate3

2-Phosphoglycerate3 34 phosphoenolpyruvate3 H2O Phosphoenolpyruvate3 ADP3 H 34 pyruvate ATP4

Pyruvate NADH H 34 lactate NAD

Hexokinase

Hexokinase

Glucokinase

Phosphoglucoisomerase

Phosphofructokinase

Fructose bisphosphate aldolase Triose phosphate isomerase Glyceraldehyde-3-P dehydrogenase Phosphoglycerate kinase Phosphoglycerate mutase

Enolase

Pyruvate kinase

Lactate dehydrogenase

continued

Table 19.2

Steady-State Concentrations of

Glycolytic Metabolites in Erythrocytes

Metabolite	mM
Glucose	5.0
Glucose-6-phosphate	0.083
Fructose-6-phosphate	0.014
Fructose-1,6-bisphosphate	0.031
Dihydroxyacetone phosphate	0.14
Glyceraldehyde-3-phosphate	0.019
1,3-Bisphosphoglycerate	0.001
2,3-Bisphosphoglycerate	4.0
3-Phosphoglycerate	0.12
2-Phosphoglycerate	0.030
Phosphoenolpyruvate	0.023
Pyruvate	0.051
Lactate	2.9
ATP	1.85
ADP	0.14
Pi	1.0

Adapted from Minakami, S., and Yoshikawa, H., 1965.

Biochemical and Biophysical Research Communications 18:345.

Equation 3.12 in Chapter 3 and the data in Table 19.2 can be used to calculate a value for G for the hexokinase, glucokinase reaction in erythrocytes:

G G° RT ln	[G-6-P][ADP]		(19.5)
	[Glu][ATP]
G 16.7 kJ/mol (8.314 J/mol			K)(310 K) ln	[0.083][0.14]
				[5.0][1.85]
G 33.9 kJ/mol

Thus, G is even more favorable under cellular conditions than at standard state. As we will see later in this chapter, the hexokinase, glucokinase reaction is one of several that drive glycolysis forward.

The Cellular Advantages of Phosphorylating Glucose

The incorporation of a phosphate into glucose in this energetically favorable reaction is important for several reasons. First, phosphorylation keeps the substrate in the cell. Glucose is a neutral molecule and could diffuse across the cell membrane, but phosphorylation confers a negative charge on glucose, and the plasma membrane is essentially impermeable to glucose-6-phosphate (Figure 19.4). Moreover, rapid conversion of glucose to glucose-6-phosphate keeps the intracellular concentration of glucose low, favoring diffusion of glucose into the cell. In addition, because regulatory control can be imposed only on reactions not at equilibrium, the favorable thermodynamics of this first reaction makes it an important site for regulation.

Hexokinase

In most animal, plant, and microbial cells, the enzyme that phosphorylates glucose is hexokinase. Magnesium ion (Mg2 ) is required for this reaction, as for the other kinase enzymes in the glycolytic pathway. The true substrate for the hexokinase reaction is MgATP2 . The apparent Km for glucose of the animal

FIGURE 19.4

19.3 ● The First Phase of Glycolysis

615

Table 19.1

Continued

	Subunit	Oligomeric	G°	Keq	G
Source	Molecular Weight (Mr)	Composition	(kJ/mol)	at 25°C	(kJ/mol)
Mammals	100,000	Monomer	16.7	850	33.9*
Yeast	55,000	Dimer
Mammalian liver	50,000	Monomer
Human	65,000	Dimer	1.67	0.51	2.92
Rabbit muscle	78,000	Tetramer	14.2	310	18.8
Rabbit muscle	40,000	Tetramer	23.9	6.43 10 5	0.23
Chicken muscle	27,000	Dimer	7.56	0.0472	2.41
Rabbit muscle	37,000	Tetramer	6.30	0.0786	1.29
Rabbit muscle	64,000	Monomer	18.9	2060	0.1
Rabbit muscle	27,000	Dimer	4.4	0.169	0.83
Rabbit muscle	41,000	Dimer	1.8	0.483	1.1
Rabbit muscle	57,000	Tetramer	31.7	3.63 105	23.0
Rabbit muscle	35,000	Tetramer	25.2	2.63 104	14.8

* G values calculated for 310 K (37°C) using the data in Table 19.2 for metabolite concentrations in erythrocytes. G° values are assumed to be the same at 25°C and 37°C.

skeletal muscle enzyme is approximately 0.1 mM, and the enzyme thus operates efficiently at normal blood glucose levels of 4 mM or so. Different body tissues possess different isozymes of hexokinase, each exhibiting somewhat different kinetic properties. The animal enzyme is allosterically inhibited by the product, glucose-6-phosphate. High levels of glucose-6-phosphate inhibit hexokinase activity until consumption by glycolysis lowers its concentration. The hexokinase reaction is one of three points in the glycolysis pathway that are regulated. As the generic name implies, hexokinase can phosphorylate a variety of hexose sugars, including glucose, mannose, and fructose.

Glucokinase

Liver contains an enzyme called glucokinase, which also carries out the reaction in Figure 19.4 but is highly specific for D-glucose, has a much higher Km for glucose (approximately 10.0 mM), and is not product-inhibited. With such

Extracellular	Cytoplasm
fluid
Glucose	Glucose

ATP

ADP

Glucose is kept in the cell	Glucose-
by phosphorylation to G6P,	6-phosphate
which cannot easily cross
the plasma membrane

● Phosphorylation of glucose to glucose-6-phosphate by ATP creates a charged molecule that cannot easily cross the plasma membrane.

616 Chapter 19 ● Glycolysis

a high Km for glucose, glucokinase becomes important metabolically only when liver glucose levels are high (for example, when the individual has consumed large amounts of sugar). When glucose levels are low, hexokinase is primarily responsible for phosphorylating glucose. However, when glucose levels are high, glucose is converted by glucokinase to glucose-6-phosphate and is eventually stored in the liver as glycogen. Glucokinase is an inducible enzyme—the amount present in the liver is controlled by insulin (secreted by the pancreas). (Patients with diabetes mellitus produce insufficient insulin. They have low levels of glucokinase, cannot tolerate high levels of blood glucose, and produce little liver glycogen.) Because glucose-6-phosphate is common to several metabolic pathways (Figure 19.5), it occupies a branch point in glucose metabolism.

Reaction 2: Phosphoglucoisomerase Catalyzes the

Isomerization of Glucose-6-Phosphate

The second step in glycolysis is a common type of metabolic reaction: the isomerization of a sugar. In this particular case, the carbonyl oxygen of glucose- 6-phosphate is shifted from C-1 to C-2. This amounts to isomerization of an aldose (glucose-6-phosphate) to a ketose—fructose-6-phosphate (Figure 19.6). The reaction is necessary for two reasons. First, the next step in glycolysis is phosphorylation at C-1, and the hemiacetal OOH of glucose would be more difficult to phosphorylate than a simple primary hydroxyl. Second, the isomerization to fructose (with a carbonyl group at position 2 in the linear form) activates carbon C-3 for cleavage in the fourth step of glycolysis. The enzyme responsible for this isomerization is phosphoglucoisomerase, also known as glucose phosphate isomerase. In humans, the enzyme requires Mg2 for activity and is highly specific for glucose-6-phosphate. The G° is 1.67 kJ/mol, and the value of G under cellular conditions (Table 19.1) is 2.92 kJ/mol. This small value means that the reaction operates near equilibrium in the cell and is readily reversible. Phosphoglucoisomerase proceeds through an enediol intermediate, as shown in Figure 19.6. Although the predominant forms of glucose- 6-phosphate and fructose-6-phosphate in solution are the ring forms (Figure 19.6), the isomerase interconverts the open-chain form of G-6-P with the openchain form of F-6-P. The first reaction catalyzed by the isomerase is the opening of the pyranose ring (Figure 19.6, Step A). In the next step, the C-2 proton is removed from the substrate by a basic residue on the enzyme, facilitating formation of the enediol intermediate (Figure 19.6, Step B). This process then

Pentose phosphate pathway

Synthesis of NADPH and 4–C, 5–C, and 7–C sugars

Glucose

Glycogen Energy storage

in liver and muscles

Glucose-6-phosphate Glucose-1-phosphate

Glucuronate Carbohydrate

synthesis

Fructose-6-phosphate Glucosamine-6-phosphate

Glycolysis continues

FIGURE 19.5 ● Glucose-6-phosphate is the branch point for several metabolic pathways.

19.3 ● The First Phase of Glycolysis

617

CH

OPO2–

.+

CH

OPO2–

2

3

H .B

E

2

3 .

O H

OH

. B

E

Step A

H

H

H

H

H

C

OH

OH H

. +

HO

O

H

HO

O

.

H . B

E

H

OH

.B

E

H

OH

Step B

CH2OPO23– .

2–

.

+

. B

E

CH

OPO

H

OH

2

3

H . B

E

Step C

OH

H

H

H

CH2OH

H

C

HO

C

C

OH

H

O

+

HO

.

H

H .B

E

O

H

.

–2O3POH2C

O

CH2OH

.B

E

H HO

H

OH

OH

FIGURE 19.6 ● The phosphoglucoisomerase mechanism involves opening of the pyranose ring (Step A), proton abstraction leading to enediol formation (Step B), and proton addition to the double bond, followed by ring closure (Step C).

operates somewhat in reverse (Figure 19.6, Step C), creating a carbonyl group at C-2 to complete the formation of fructose-6-phosphate. The furanose form of the product is formed in the usual manner by attack of the C-5 hydroxyl on the carbonyl group, as shown.

Reaction 3: Phosphofructokinase—The Second Priming Reaction

The action of phosphoglucoisomerase, “moving” the carbonyl group from C-1 to C-2, creates a new primary alcohol function at C-1 (see Figure 19.5). The next step in the glycolytic pathway is the phosphorylation of this group by phosphofructokinase. Once again, the substrate that provides the phosphoryl group is ATP. Like the hexokinase, glucokinase reaction, the phosphorylation of fructose-6-phosphate is a priming reaction and is endergonic:

Fructose-6-P Pi 88n fructose-1,6-bisphosphate	(19.6)
G° 16.3 kJ/mol

When coupled (by phosphofructokinase) with the hydrolysis of ATP, the overall reaction (Figure 19.7) is strongly exergonic:

Fructose-6-P ATP 88n fructose-1,6-bisphosphate ADP (19.7)G° 14.2 kJ/mol

G (in erythrocytes) 18.8 kJ/mol

At pH 7 and 37°C, the phosphofructokinase reaction equilibrium lies far to the right. Just as the hexokinase reaction commits the cell to taking up glucose, the phosphofructokinase reaction commits the cell to metabolizing glucose rather than converting it to another sugar or storing it. Similarly, just as the large free energy change of the hexokinase reaction makes it a likely candidate for regulation, so the phosphofructokinase reaction is an important site of regulation— indeed, the most important site in the glycolytic pathway.

Phosphofructokinase with ADP shown in white and fructose-6-P in red.

FIGURE 19.8

618 Chapter 19 ● Glycolysis

O23–P OCH2
				O23–P OCH2	O	CH2O	PO23–
O		CH2OH	Mg2+
								+ ADP
H	HO	+ ATP	Phosphofructokinase	H	HO
H		OH		H		OH
			(PFK)
OH	H			OH H
Fructose-6-phosphate			∆ G ' = –14.2 kJ/mol	Fructose-1,6-bisphosphate

∆ Gerythrocyte = –18.8 kJ/mol

FIGURE 19.7 ● The phosphofructokinase reaction.

velocity		Low [ATP]
Reaction		High [ATP]

[Fructose-6-phosphate]

● At high [ATP], phosphofructokinase (PFK) behaves cooperatively, and the plot of enzyme activity versus [fructose-6-phos- phate] is sigmoid. High [ATP] thus inhibits PFK, decreasing the enzyme’s affinity for fruc- tose-6-phosphate.

Regulation of Phosphofructokinase

Phosphofructokinase is the “valve” controlling the rate of glycolysis. ATP is an allosteric inhibitor of this enzyme. In the presence of high ATP concentrations, phosphofructokinase behaves cooperatively, plots of enzyme activity versus fructose-6-phosphate are sigmoid, and the Km for fructose-6-phosphate is increased (Figure 19.8). Thus, when ATP levels are sufficiently high in the cytosol, glycolysis “turns off.” Under most cellular conditions, however, the ATP concentration does not vary over a large range. The ATP concentration in muscle during vigorous exercise, for example, is only about 10% lower than that during the resting state. The rate of glycolysis, however, varies much more. A large range of glycolytic rates cannot be directly accounted for by only a 10% change in ATP levels.

AMP reverses the inhibition due to ATP, and AMP levels in cells can rise dramatically when ATP levels decrease, due to the action of the enzyme adenylate kinase, which catalyzes the reaction

ADP ADP 34 ATP AMP

	with the equilibrium constant:
	Keq	[ATP][AMP]	0.44	(19.8)
		[ADP]2

Adenylate kinase rapidly interconverts ADP, ATP, and AMP to maintain this equilibrium. ADP levels in cells are typically 10% of ATP levels, and AMP levels are often less than 1% of the ATP concentration. Under such conditions, a small net change in ATP concentration due to ATP hydrolysis results in a much larger relative increase in the AMP levels because of adenylate kinase activity.

EXAMPLE

Calculate the change in concentration in AMP that would occur if 8% of the ATP in an erythrocyte (red blood cell) were suddenly hydrolyzed to ADP. In erythrocytes (Table 19.2), the concentration of ATP is typically 1850 M, the concentration of ADP is 145 M, and the concentration of AMP is 5 M. The total adenine nucleotide concentration is 2000 M.

SOLUTION

The problem can be solved using the equilibrium expression for the adenylate kinase reaction:

[ATP][AMP] Keq 0.44 [ADP]2

FIGURE 19.10

If 8% of the ATP is hydrolyzed to ADP, then [ATP] becomes 1850(0.92) 1702 M, and [AMP] [ADP] becomes 2000 1702 298 M, and [AMP] may be calculated from the adenylate kinase equilibrium:

	[1702
0.44		M][AMP]

[ADP]2

Since [AMP] 298 M [ADP],

0.44

1702(298 [ADP])

[ADP]2

[ADP] 278 M

[AMP] 20 M

Thus, an 8% decrease in [ATP] results in a 20/5 or fourfold increase in the concentration of AMP.

Clearly, the activity of phosphofructokinase depends both on ATP and AMP levels and is a function of the cellular energy status. Phosphofructokinase activity is increased when the energy status falls and is decreased when the energy status is high. The rate of glycolysis activity thus decreases when ATP is plentiful and increases when more ATP is needed.

Glycolysis and the citric acid cycle (to be discussed in Chapter 20) are coupled via phosphofructokinase, because citrate, an intermediate in the citric acid cycle, is an allosteric inhibitor of phosphofructokinase. When the citric acid cycle reaches saturation, glycolysis (which “feeds” the citric acid cycle under aerobic conditions) slows down. The citric acid cycle directs electrons into the electron transport chain (for the purpose of ATP synthesis in oxidative phosphorylation) and also provides precursor molecules for biosynthetic pathways. Inhibition of glycolysis by citrate ensures that glucose will not be committed to these activities if the citric acid cycle is already saturated.

Phosphofructokinase is also regulated by -D-fructose-2,6-bisphosphate, a potent allosteric activator that increases the affinity of phosphofructokinase for the substrate fructose-6-phosphate (Figure 19.9). Stimulation of phosphofructokinase is also achieved by decreasing the inhibitory effects of ATP (Figure 19.10). Fructose-2,6-bisphosphate increases the net flow of glucose through glycolysis by stimulating phosphofructokinase and, as we shall see in Chapter 23, by inhibiting fructose-1,6-bisphosphatase, the enzyme that catalyzes this reaction in the opposite direction.

Reaction 4: Cleavage of Fructose-1,6-bisP by

Fructose Bisphosphate Aldolase

Fructose bisphosphate aldolase cleaves fructose-1,6-bisphosphate between the C-3 and C-4 carbons to yield two triose phosphates. The products are dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate. The reaction (Figure 19.11) has an equilibrium constant of approximately 10 4 M, and a corresponding G° of 23.9 kJ/mol. These values might imply that the reaction does not proceed effectively from left to right as written. However, the reaction makes two molecules (glyceraldehyde-3-P and dihydroxyacetone-P) from one molecule (fructose-1,6-bisphosphate), and the equilibrium is thus greatly influenced by concentration. The value of G in erythrocytes is actually 0.23 kJ/mol (see Table 19.1). At physiological concentrations, the reaction is essentially at equilibrium.

19.3 ● The First Phase of Glycolysis

619

1.0 M F–2,6–BP

100

velocity	80
				0.1	M
Relative	60
	40
	20		0
	0		1		2		3	4	5
			[Fructose-6-phosphate] ( M)

FIGURE 19.9 ● Fructose-2,6-bisphosphate activates phosphofructokinase, increasing the affinity of the enzyme for fructose-6-phosphate and restoring the hyperbolic dependence of enzyme activity on substrate.

1.0 M F–2,6–BP

0.1 M

	velocity	0
	Relative

0	1	2	3	4	5
			[ATP] ( M)

● Fructose-2,6-bisphosphate decreases the inhibition of phosphofructokinase due to ATP.

2–O3POCH2				O		OPO32–
	H			HO
H						CH2OH

OH H

Fructose-2,6-bisphosphate

620 Chapter 19 ● Glycolysis

H	H	O–
C
	O
R H	O–	C	O
	R	H H	R'
H	R'

R'= H (aldehyde)

R'= alkyl, etc. (ketone)

FIGURE 19.12 ● An aldol condensation reaction.

Triose phosphate isomerase with substrate analog 2-phosphoglycerate shown in red.

CH2O

PO23–

O

C

Fructose

H

O

CH2O

PO23–

Aldol

HO

C

H

bisphosphate

C

aldolase

O

C

+

cleavage H

C

OH

H

C

OH

H

C

OH

CH2OH

CH2O

PO23–

CH2O

PO23–

D-Fructose-1,6-bisphosphate

Dihydroxyacetone

D-Glyceraldehyde

(FBP)

phosphate (DHAP)

3-phosphate (G-3-P)

∆ G°' = 23.9 kJ/mol

FIGURE 19.11 ● The fructose-1,6-bisphosphate aldolase reaction.

Two classes of aldolase enzymes are found in nature. Animal tissues produce a Class I aldolase, characterized by the formation of a covalent Schiff base intermediate between an active-site lysine and the carbonyl group of the substrate. Class I aldolases do not require a divalent metal ion (and thus are not inhibited by EDTA) but are inhibited by sodium borohydride, NaBH4, in the presence of substrate (see A Deeper Look, page 622). Class II aldolases are produced mainly in bacteria and fungi and are not inhibited by borohydride, but do contain an active-site metal (normally zinc, Zn2 ) and are inhibited by EDTA. Cyanobacteria and some other simple organisms possess both classes of aldolase.

The aldolase reaction is merely the reverse of the aldol condensation well known to organic chemists. The latter reaction involves an attack by a nucleophilic enolate anion of an aldehyde or ketone on the carbonyl carbon of an aldehyde (Figure 19.12). The opposite reaction, aldol cleavage, begins with removal of a proton from the -hydroxyl group, which is followed by the elimination of the enolate anion. A mechanism for the aldol cleavage reaction of fructose-1,6-bisphosphate in the Class I–type aldolases is shown in Figure 19.13a. In Class II aldolases, an active-site metal such as Zn2 behaves as an electrophile, polarizing the carbonyl group of the substrate and stabilizing the enolate intermediate (Figure 19.13b).

Reaction 5: Triose Phosphate Isomerase

Of the two products of the aldolase reaction, only glyceraldehyde-3-phosphate goes directly into the second phase of glycolysis. The other triose phosphate, dihydroxyacetone phosphate, must be converted to glyceraldehyde-3-phos- phate by the enzyme triose phosphate isomerase (Figure 19.14). This reaction thus permits both products of the aldolase reaction to continue in the glycolytic pathway, and in essence makes the C-1, C-2, and C-3 carbons of the starting glucose molecule equivalent to the C-6, C-5, and C-4 carbons, respectively. The reaction mechanism involves an enediol intermediate that can donate either of its hydroxyl protons to a basic residue on the enzyme and thereby become either dihydroxyacetone phosphate or glyceraldehyde-3-phosphate (Figure 19.15). Triose phosphate isomerase is one of the enzymes that have evolved to a state of “catalytic perfection,” with a turnover number near the diffusion limit (Chapter 14, Table 14.5).

The triose phosphate isomerase reaction completes the first phase of glycolysis, each glucose that passes through being converted to two molecules of glyceraldehyde-3-phosphate. Although the last two steps of the pathway are