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

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The Phosphorylase Cascade Amplifies the Hormonal Signal

Stimulation of glycogen breakdown involves consumption of molecules of ATP at three different steps in the hormone-sensitive adenylyl cyclase cascade (Figure 15.19). Note that the cascade mechanism is a means of chemical amplification, because the binding of just a few molecules of epinephrine or glucagon results in the synthesis of many molecules of cyclic AMP, which, through the action of cAMP-dependent protein kinase, can activate many more molecules of phosphorylase kinase and even more molecules of phosphorylase. For example, an extracellular level of 10 10 to 10 8 M epinephrine prompts the formation of 10 6 M cyclic AMP, and for each protein kinase activated by cyclic AMP, approximately 30 phosphorylase kinase molecules are activated; these in turn activate some 800 molecules of phosphorylase. Each of these catalyzes the formation of many molecules of glucose-1-P.

The Difference Between Epinephrine and Glucagon

Although both epinephrine and glucagon exert glycogenolytic effects, they do so for quite different reasons. Epinephrine is secreted as a response to anger or fear and may be viewed as an alarm or danger signal for the organism. Called the “fight or flight” hormone, it prepares the organism for mobilization of large amounts of energy. Among the many physiological changes elicited by epinephrine, one is the initiation of the enzyme cascade, as in Figure 15.19, which leads to rapid breakdown of glycogen, inhibition of glycogen synthesis, stimulation of glycolysis, and production of energy. The burst of energy produced is the result of a 2000-fold amplification of the rate of glycolysis. Because a fear or anger response must include generation of energy (in the form of glucose) —both immediately in localized sites (the muscles) and eventually throughout the organism (as supplied by the liver)—epinephrine must be able to activate glycogenolysis in both liver and muscles.

Glucagon is involved in the long-term maintenance of steady-state levels of glucose in the blood and other tissues. It performs this function by stimulating the liver to release glucose from glycogen stores into the bloodstream. To further elevate glucose levels, glucagon also activates liver gluconeogenesis. It is important to note, however, that stabilization of blood glucose levels is managed almost entirely by the liver. Glucagon does not activate the phosphorylase cascade in muscle (muscle membranes do not contain glucagon receptors). Muscle glycogen breakdown occurs only in response to epinephrine release, and muscle tissue does not participate in maintenance of steady-state glucose levels in the blood.

Cortisol and Glucocorticoid Effects on Glycogen Metabolism

Glucocorticoids are a class of steroid hormones that exert distinct effects on liver, skeletal muscle, and adipose tissue. The effects of cortisol, a typical glucocorticoid, are best described as catabolic because cortisol promotes protein breakdown and decreases protein synthesis in skeletal muscle. In the liver, however, it stimulates gluconeogenesis and increases glycogen synthesis. Cortisolinduced gluconeogenesis results primarily from increased conversion of amino acids into glucose (Figure 23.25). Specific effects of cortisol in the liver include increased gene expression of several of the enzymes of the gluconeogenic pathway, activation of enzymes involved in amino acid metabolism, and stimulation of the urea cycle, which disposes of nitrogen liberated during amino acid catabolism (Chapter 27).

23.5 ● Control of Glycogen Metabol

H+3N His Ser Glu Gly Th

Tyr Lys Ser Ty

Leu

Asp

Ser Arg Arg Ala

Leu Trp Gln

Met

Asn

Thr COO–

FIGURE 23.23 ● The amino acid glucagon.

OH

OH

HO C H

CH2

NH+2

CH3

FIGURE 23.24 ● Epinephrine

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762 Chapter 23 ● Gluconeogenesis, Glycogen Metabolism, and the Pentose Phosphate Pathway

FIGURE 23.25 ● The effects of cortisol on carbohydrate and protein metabolism in the liver.

	Plasma		Liver
					Urea
Amino acids			Amino acid	Nitrogen	cycle
			metabolizing
			enzymes
				+	+
		Gluconeogenesis			Urea
Glucose				+
		Glycogen	+
		synthesis
				Cortisol

23.6 ● The Pentose Phosphate Pathway

Cells require a constant supply of NADPH for reductive reactions vital to biosynthetic purposes. Much of this requirement is met by a glucose-based metabolic sequence variously called the pentose phosphate pathway, the hexose monophosphate shunt, or the phosphogluconate pathway. In addition to providing NADPH for biosynthetic processes, this pathway produces ribose-5-phosphate, which is essential for nucleic acid synthesis. Several metabolites of the pentose phosphate pathway can also be shuttled into glycolysis.

An Overview of the Pathway

The pentose phosphate pathway begins with glucose-6-phosphate, a six-carbon sugar, and produces three-, four-, five-, six-, and seven-carbon sugars (Figure 23.26). As we will see, two successive oxidations lead to the reduction of NADP to NADPH and the release of CO2. Five subsequent nonoxidative steps produce a variety of carbohydrates, some of which may enter the glycolytic pathway. The enzymes of the pentose phosphate pathway are particularly abundant in the cytoplasm of liver and adipose cells. These enzymes are largely absent in muscle, where glucose-6-phosphate is utilized primarily for energy production via glycolysis and the TCA cycle. These pentose phosphate pathway enzymes are located in the cytosol, which is the site of fatty acid synthesis, a pathway heavily dependent on NADPH for reductive reactions.

The Oxidative Steps of the Pentose Phosphate Pathway

(1) Glucose-6-Phosphate Dehydrogenase

The pentose phosphate pathway begins with the oxidation of glucose-6-phos- phate. The products of the reaction are a cyclic ester (the lactone of phosphogluconic acid) and NADPH (Figure 23.27). Glucose-6-phosphate dehydrogenase, which catalyzes this reaction, is highly specific for NADP . As the first step of a major pathway, the reaction is irreversible and highly regulated. Glucose-6-phosphate dehydrogenase is strongly inhibited by the product coenzyme, NADPH, and also by fatty acid esters of coenzyme A (which are

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23.6 ● The Pentose Phosphate Path

Reductive anabolic pathways

NADPH

NADPH

+ H+

+

H+

O

O

O–

C

H

NADP+

C

NADP+

H

H

C

OH

H

C

OH

H

C

OH

H2O

CO2

HO

C

H

HO

C

H

C

O

1 , 2

3

H

C

OH

H

C

OH

H

C

OH

H

C

OH

H

C

OH

H

C

OH

H2C

O

P

H2C

O

P

H2C

O

P

Glucose-6-

6-Phospho-

Ribulose-5-

phosphate

gluconate

phosphate

Nucleic acid

H

biosynthesis

H

C

OH

O

C

O

C

H

HO

C

H

H

C

OH

H

C

OH

H

C

OH

H

C

OH

H

H

C

OH

H

C

OH

H C

O

P

H C

O

P

H

C

OH

4

2

2

C

O

Ribose-5-

Sedoheptulose-

H

C

OH

phosphate

7-phosphate

H

C

OH

H

6

7

H2C

O

P

5

H

C

OH

O

Ribulose-5-

C

O

phosphate

HO

C

H

C

H

H

C

OH

H

C

OH

H2C

O

P

H2C

O

P

Garrett & Grisham

Xylulose-5-

Glyceraldehyde-

Biochemistry 2/e

phosphate

3-phosphate

Figure 23.26 42p6 x 54p

FIGURE5/4/9823.26 ● The pentose phosphate pathway. The numerals in the blue circles indicate the steps discussed in the text.

O

C H

H C OH

H C OH

H2C

O

P

Another

Erythrose-4-

Xylulose-5-

phosphate

phosphate

H

8

H

C

OH

C

O

O

H

HO

C

H

C

OH

C

H

H

C

OH

H

C

O

H2C

O

P

H2C

O

Fructose-6-

Glyceraldehyd

phosphate

3-phosphate

Glycolytic intermediates

FIGURE 23.27

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764 Chapter 23 ● Gluconeogenesis, Glycogen Metabolism, and the Pentose Phosphate Pathway

● The glucose-6-phosphate dehydrogenase reaction is the committed step in the pentose phosphate pathway.

Step 1
	2–O	POCH		2
3						O
		OH									α -D-Glucose-6-phosphate
	HO						OH
						OH
	NADP+					Glucose-6-P dehydrogenase
NADPH + H+
	2–O	POCH		2
3						O
		OH							O		6-Phospho-D-gluconolactone

HO

OH

intermediates of fatty acid biosynthesis). Inhibition due to NADPH depends upon the cytosolic NADP /NADPH ratio, which in the liver is about 0.015 (compared to about 725 for the NAD /NADH ratio in the cytosol).

(2) Gluconolactonase

The gluconolactone produced in step 1 is hydrolytically unstable and readily undergoes a spontaneous ring-opening hydrolysis, although an enzyme, gluconolactonase, accelerates this reaction (Figure 23.28). The linear product, the sugar acid 6-phospho-D-gluconate, is further oxidized in step 3.

(3) 6-Phosphogluconate Dehydrogenase

The oxidative decarboxylation of 6-phosphogluconate by 6-phosphogluconate dehydrogenase yields D-ribulose-5-phosphate and another equivalent of NADPH. There are two distinct steps in this reaction (Figure 23.29): the initial NADP -dependent dehydrogenation yields a -keto acid, 3-keto-6-phos- phogluconate, which is very susceptible to decarboxylation (the second step). The resulting product, D-ribulose-5-P, is the substrate for the nonoxidative reactions composing the rest of this pathway.

The Nonoxidative Steps of the Pentose Phosphate Pathway

This portion of the pathway begins with an isomerization and an epimerization, and it leads to the formation of either D-ribose-5-phosphate or D-xylulose- 5-phosphate. These intermediates can then be converted into glycolytic intermediates or directed to biosynthetic processes.

									COO–
FIGURE 23.28 ● The gluconolactonase reac-	Step 2
tion.	2–O3POCH2								HCOH
				O			Gluconolactonase
						O			HOCH
			OH
	HO								HCOH
				OH			H2O	H+
									HCOH
	6-P-D-Gluconolactone
									CH2OPO23–
									6-P-D-Gluconate

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Step 3

COO–

H+

COO–

+

CH2OH

HCOH

HCOH

NADP+ NADPH

H+

CO2

O

HOCH

C

O

C

HCOH

6-Phosphogluconate

HCOH

HCOH

dehydrogenase

HCOH

HCOH

HCOH

CH2OPO 32 –

CH2OPO 32 –

CH2OPO 32 –

6-P-D-Gluconate

3-keto-6-P-D-Gluconate

D-Ribulose-5-P

(4) Phosphopentose Isomerase

This enzyme interconverts ribulose-5-P and ribose-5-P via an enediol intermediate (Figure 23.30). The reaction (and mechanism) is quite similar to the phosphoglucoisomerase reaction of glycolysis, which interconverts glucose-6-P and fructose-6-P. The ribose-5-P produced in this reaction is utilized in the biosynthesis of coenzymes (including NADH, NADPH, FAD, and B12), nucleotides, and nucleic acids (DNA and RNA). The net reaction for the first four steps of the pentose phosphate pathway is

Glucose-6-P 2 NADP 88n ribose-5-P 2 NADPH 2 H CO2

(5) Phosphopentose Epimerase

This reaction converts ribulose-5-P to another ketose, namely, xylulose-5-P. This reaction also proceeds by an enediol intermediate, but involves an inversion at C-3 (Figure 23.31). In the reaction, an acidic proton located - to a carbonyl carbon is removed to generate the enediolate, but the proton is added back to the same carbon from the opposite side. Note the distinction in nomenclature here. Interchange of groups on a single carbon is an epimerization, and interchange of groups between carbons is referred to as an isomerization.

To this point, the pathway has generated a pool of pentose phosphates. The G° for each of the last two reactions is small, and the three pentose-5- phosphates coexist at equilibrium. The pathway has also produced two molecules of NADPH for each glucose-6-P converted to pentose-5-phosphate. The next three steps rearrange the five-carbon skeletons of the pentoses to produce three-, four-, six-, and seven-carbon units, which can be used for various metabolic purposes. Why should the cell do this? Very often, the cellular need for

Step 4

CH2OH

HC

OH

HC

O

C

O

HCOH

C

OH

HCOH

HCOH

HCOH

HCOH

HCOH

HCOH

CH2OPO23–

CH2OPO23–

CH2OPO23–

D-Ribulose-5-P (ketose)

Enediol

Ribose-5-P (aldose)

23.6 ● The Pentose Phosphate Path

FIGURE 23.29 ● The 6-phosphog dehydrogenase reaction.

FIGURE 23.30 ● The phosphope merase reaction involves an enediol intermediate.

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766

Chapter 23 ●

Gluconeogenesis, Glycogen Metabolism, and the Pentose Phosphate Pathway

Step 5

CH2OH

CH2OH

CH2OH

Phosphopentose

O–

C

O

C

C

O

epimerase

HB+

E

E

B

H

COH

C

OH

HO

C

H

H

COH

HCOH

H COH

CH2OPO23–

CH2OPO23–

CH2OPO23–

Ribulose-5-P

Enediolate

Xylulose-5-P

FIGURE 23.31 ● The phosphopentose epimerase reaction interconverts ribulose-5-P and xylulose-5-phosphate. The mechanism involves an enediol intermediate and occurs with inversion at C-3.

NADPH is considerably greater than the need for ribose-5-phosphate. The next three steps thus return some of the five-carbon units to glyceraldehyde-3-phos- phate and fructose-6-phosphate, which can enter the glycolytic pathway. The advantage of this is that the cell has met its needs for NADPH and ribose-5- phosphate in a single pathway, yet at the same time it can return the excess carbon metabolites to glycolysis.

(6) and (8) Transketolase

The transketolase enzyme acts at both steps 6 and 8 of the pentose phosphate pathway. In both cases, the enzyme catalyzes the transfer of two-carbon units. In these reactions (and also in step 7, the transaldolase reaction, which transfers three-carbon units), the donor molecule is a ketose and the recipient is an aldose. In step 6, xylulose-5-phosphate transfers a two-carbon unit to ribose-5-phosphate to form glyceraldehyde-3-phosphate and sedoheptulose- 7-phosphate (Figure 23.32). Step 8 involves a two-carbon transfer from xylu- lose-5-phosphate to erythrose 4-phosphate to produce another glyceraldehyde- 3-phosphate and a fructose-6-phosphate (Figure 23.33). Three of these products enter directly into the glycolytic pathway. (The sedoheptulose-7-phosphate is taken care of in step 7, as we shall see.) Transketolase is a thiamine pyrophos- phate–dependent enzyme, and the mechanism (Figure 23.34) involves abstraction of the acidic thiazole proton of TPP, attack by the resulting carbanion at the carbonyl carbon of the ketose phosphate substrate, expulsion of the glyc- eraldehyde-3-phosphate product, and transfer of the two-carbon unit. Transketolase can process a variety of 2-keto sugar phosphates in a similar manner. It is specific for ketose substrates with the configuration shown, but can accept a variety of aldose phosphate substrates.

CH2OH

FIGURE 23.32

● The transketolase reaction

Step 6

of step 6 in the pentose phosphate pathway.

C

O

CH2OH

CHO

HOCH

O

CHO

C

HCOH

HCOH

+

Transketolase

+

HOCH

HCOH

HCOH

HCOH

CH2OPO32–

HCOH

HCOH

HCOH

CH2OPO23–

CH2OPO23–

CH2OPO23–

Xylulose-5-P

Ribose-5-P

Glyceraldehyde-3-P

Sedoheptulose-7-P

FIGURE 23.34

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23.6 ● The Pentose Phosphate Path

FIGURE 23.33 ● The transketolas

Step 8

CH2OH

of step 8 in the pentose phosphate

CH2OH

CHO

C

O

C

O

CHO

HOCH

+

Transketolase

+

HOCH

HCOH

HCOH

HCOH

CH2OPO32–

HCOH

HCOH

HCOH

CH2OPO32–

CH2OPO32–

CH2OPO32–

Xylulose-5-P

Erythrose-4-P

Glyceraldehyde-3-P

Fructose-6-P

	S	CH2OH			S			CH2OH
R				R
		–						C	OH
	+		O		+
		C
	N				N	H	O	CH

R'	HOCH		R'
	R"		R"
								HCOH
	HCOH			E		B
								CH2OPO23–
	CH2OPO32–
	D–Xylulose-5-P
			CHO
			Glyceraldehyde HCOH
			CH2OPO23–

	S	CH2OH
R
		C	OH
	+	–
	N

R'

R"

HC O	H B E
HCOH
HCOH
HCOH
CH2OPO32–

	R	S	CH2OH
			C
		N	OH
		R'
		R"

				D–Ribose-5-P
R	S	CH2OH				CH2OH
	–			H+
	+	+	O	R	S	C	O	H
	N	C			+
	R'
		HOCH			N HOCH
	R"
					R'
		HCOH			R''	HCOH

HCOH	HCOH
HCOH	HCOH
CH2OPO32–	CH2OPO32–

Sedoheptulose-7-P

● The mechanism dependent transketolase reaction. I the group transferred in the transk tion might best be described as an a whereas the transferred group in th dolase reaction is actually a ketol. D irony, these names persist for histor

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768Chapter 23 ● Gluconeogenesis, Glycogen Metabolism, and the Pentose Phosphate Pathway

(7)Transaldolase

The transaldolase functions primarily to make a useful glycolytic substrate from the sedoheptulose-7-phosphate produced by the first transketolase reaction. This reaction (Figure 23.35) is quite similar to the aldolase reaction of glycolysis, involving formation of a Schiff base intermediate between the sedohep- tulose-7-phosphate and an active-site lysine residue (Figure 23.36). Elimination of the erythrose-4-phosphate product leaves an enamine of dihydroxyacetone, which remains stable at the active site (without imine hydrolysis) until the other substrate comes into position. Attack of the enamine carbanion at the carbonyl carbon of glyceraldehyde-3-phosphate is followed by hydrolysis of the Schiff base (imine) to yield the product fructose-6-phosphate.

Utilization of Glucose-6-P Depends on the Cell’s

Need for ATP, NADPH, and Ribose-5-P

It is clear that glucose-6-phosphate can be used as a substrate either for glycolysis or for the pentose phosphate pathway. The cell makes this choice on the basis of its relative needs for biosynthesis and for energy from metabolism. ATP can be produced in abundance if glucose-6-phosphate is channeled into glycolysis. On the other hand, if NADPH or ribose-5-phosphate is needed, glu- cose-6-phosphate can be directed to the pentose phosphate pathway. The molecular basis for this regulatory decision depends on the enzymes that metabolize glucose-6-phosphate in glycolysis and the pentose phosphate pathway. In glycolysis, phosphoglucoisomerase converts glucose-6-phosphate to fructose-6- phosphate, which is utilized by phosphofructokinase (a highly regulated enzyme) to produce fructose-1,6-bisphosphate. In the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (also highly regulated) produces gluconolactone from glucose-6-phosphate. Thus, the fate of glucose-6-phosphate is determined to a large extent by the relative activities of phosphofructokinase and glucose-6-P dehydrogenase. Recall (Chapter 19) that PFK is inhibited when the ATP/AMP ratio increases, and that it is inhibited by citrate but activated by fructose-2,6-bisphosphate. Thus, when the energy charge is high, glycolytic flux decreases. Glucose-6-P dehydrogenase, on the other hand, is inhibited by high levels of NADPH and also by the intermediates of fatty acid biosynthesis. Both of these are indicators that biosynthetic demands have been satisfied. If that is the case, glucose-6-phosphate dehydrogenase and the pen-

FIGURE 23.35 ● The transaldolase reaction. Step 7

CH2OH

CHO

C

O

CH2OH

CHO

HCOH

C

O

HO

CH

+

+

HCOH

HCOH

HCOH

HOCH

CH2OPO23–

CH2OPO23–

HCOH

HCOH

HCOH

HCOH

CH2OPO23–

CH2OPO23–

Sedoheptulose-7-P

Glyceraldehyde-3-P

Erythrose-4-P

Fructose-6-P

FIGURE 23.36

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23.6 ● The Pentose Phosphate Path

CH2OH

+

CH2OH

E

E

Lys

NH2

C

O

N

C

H

HO

C

H

HO

C

H

H

C

OH

H

C

O

H

R

R

RCHO (Erythrose-4-P)

CH2OH

CH2OH

CH2OH

E

+

E

H

C

C

N

N

E

H

N

C

HO

C

H

C

–

HO

H

HO

C

H

H

Enamine

H

C

OH

O

C

H

C

OH

H

C

OH

CH2OPO23–

2–

CH2OPO3

Glyceraldehyde-3-P

H2O

CH2OH

C O

HO C H

H C OH

H C OH

CH2OPO23–

Fructose--6-P

● The transaldolas nism involves attack on the substrat active-site lysine. Departure of eryth leaves the reactive enamine, which aldehyde carbon of glyceraldehydebase hydrolysis yields the second pr fructose-6-P.

tose phosphate pathway are inhibited. If NADPH levels drop, the pentose phosphate pathway turns on, and NADPH and ribose-5-phosphate are made for biosynthetic purposes.

Even when the latter choice has been made, however, the cell must still be “cognizant” of the relative needs for ribose-5-phosphate and NADPH (as well as ATP). Depending on these relative needs, the reactions of glycolysis and the pentose phosphate pathway can be combined in novel ways to emphasize the synthesis of needed metabolites. There are four principal possibilities.

(1) BOTH RIBOSE-5-P AND NADPH ARE NEEDED BY THE CELL In this case, the first four reactions of the pentose phosphate pathway predominate (Figure 23.37). NADPH is produced by the oxidative reactions of the pathway, and ribose-5-P is the principal product of carbon metabolism. As stated earlier, the net reaction for these processes is

Glucose-6-P 2 NADP H2O 88n ribose-5-P CO2 2 NADPH 2 H

(2) MORE RIBOSE-5-P THAN NADPH IS NEEDED BY THE CELL Synthesis of ribose- 5-P can be accomplished without production of NADPH if the oxidative steps of the pentose phosphate pathway are bypassed. The key to this route is the extrac-

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770 Chapter 23 ● Gluconeogenesis, Glycogen Metabolism, and the Pentose Phosphate Pathway

NADP+			NADP+		NADPH
	NADPH
Glucose-6-P		6-Phosphogluconate				Ribulose-5-phosphate		Ribose-5-phosphate
	Reactions 1,2		3				4

FIGURE 23.37 ● When biosynthetic demands dictate, the first four reactions of the pentose phosphate pathway predominate and the principal products are ribose-5-P and NADPH.

tion of fructose-6-P and glyceraldehyde-3-P, but not glucose-6-P, from glycolysis (Figure 23.38). The action of transketolase and transaldolase on fructose-6-P and glyceraldehyde-3-P produces three molecules of ribose-5-P from two molecules of fructose-6-P and one of glyceraldehyde-3-P. In this route, as in case 1, no carbon metabolites are returned to glycolysis. The net reaction for this route is

5 Glucose-6-P ATP 88n 6 ribose-5-P ADP H

(3) MORE NADPH THAN RIBOSE-5-P IS NEEDED BY THE CELL Large amounts of NADPH can be supplied for biosynthesis without concomitant production of ribose-5-P, if ribose-5-P produced in the pentose phosphate pathway is recycled to produce glycolytic intermediates. As shown in Figure 23.39, this alternative involves a complex interplay between the transketolase and transaldolase reac-

Glucose-6-P
Fructose-6-P
ATP
Fructose-1,6-bisP
DHAP Glyceraldehyde-3-P
TK
Xylulose-5-P	Erythrose-4-P
		TA
Sedoheptulose-7-P		Glyceraldehyde-3-P
Ru-5-P		TK
3-Epimerase
Ribulose-5-P	Xylulose-5-P	Ribose-5-P

Ru-5-P Isomerase

Ribose-5-P

FIGURE 23.38 ● The oxidative steps of the pentose phosphate pathway can be bypassed if the primary need is for ribose-5-P.