Dehydrogenase reaction mechanisms

FIGURE 19.18 A mechanism for the glycer-aldehyde-3-phosphate dehydrogenase reaction. Reaction of an enzyme snlfliydryl with the carbonyl carbon of glyceraldehyde-3-P forms a thiohemiacetal, which loses a hydride to NAD to become a thloester. Phosphorolysls of this thloester releases 1,3-blsphosphoglycerate. 

The pyruvate dehydrogenase complex (PDC) is a noncovalent assembly of three different enzymes operating in concert to catalyze successive steps in the conversion of pyruvate to acetyl-CoA. The active sites of ail three enzymes are not far removed from one another, and the product of the first enzyme is passed directly to the second enzyme and so on, without diffusion of substrates and products through the solution. The overall reaction (see A Deeper Look Reaction Mechanism of the Pyruvate Dehydrogenase Complex ) involves a total of five coenzymes thiamine pyrophosphate, coenzyme A, lipoic acid, NAD+, and FAD. 

The mechanism of the pyruvate dehydrogenase reaction is a tour de force of mechanistic chemistry, involving as it does a total of three enzymes (a) and five different coenzymes—thiamine pyrophosphate, lipoic acid, coenzyme A, FAD, and NAD (b). 

The first step of the u-ketoglntarate dehydrogenase reaction involves decarboxylation of the substrate and leaves a covalent TPP intermediate. Write a reasonable mechanism for this reaction. 

Based on the action of thiamine pyrophosphate in catalysis of the pyruvate dehydrogenase reaction, suggest a suitable chemical mechanism for the pyruvate decarboxylase reaction in yeast ... 

Figure 8-11. Representations of three classes of Bi-Bi reaction mechanisms. Horizontal lines represent the enzyme. Arrows indicate the addition of substrates and departure of products. Top An ordered Bi-Bi reaction, characteristic of many NAD(P)H-dependent oxidore-ductases. Center A random Bi-Bi reaction, characteristic of many kinases and some dehydrogenases. Bottom A ping-pong reaction, characteristic of aminotransferases and serine proteases.

We saw in Chapter 3 that bisubstrate reactions can conform to a number of different reaction mechanisms. We saw further that the apparent value of a substrate Km (KT) can vary with the degree of saturation of the other substrate of the reaction, in different ways depending on the mechanistic details. Hence the determination of balanced conditions for screening of an enzyme that catalyzes a bisubstrate reaction will require a prior knowledge of reaction mechanism. This places a necessary, but often overlooked, burden on the scientist to determine the reaction mechanism of the enzyme before finalizing assay conditions for HTS purposes. The importance of this mechanistic information cannot be overstated. We have already seen, in the examples of methotrexate inhibition of dihydrofolate, mycophenolic acid inhibiton of IMP dehydrogenase, and epristeride inhibition of steroid 5a-reductase (Chapter 3), how the [5]/A p ratio can influence one s ability to identify uncompetitive inhibitors of bisubstrate reactions. We have also seen that our ability to discover uncompetitive inhibitors of such reactions must be balanced with our ability to discover competitive inhibitors as well. 

Figure 5.11 Mechanism of the glyceraldehyde-3 -phosphate dehydrogenase reaction. (From Voet and Voet, 2004. Reproduced with permission from John Wiley Sons., Inc.)...

Betz, G., Warren, J. C. Reaction mechanism and stereospecificity of 20 jS-hydroxysteroid dehydrogenase. Arch. Biochem, Biophys. 128, 745—752 (1968). 

Table 11.6 Observed and intrinsic kinetic isotope effects on the glucose-6-phosphate dehydrogenase reaction in D2O (Cleland, W. W. in Cook, P. F., Ed., Enzyme Mechanism from Isotope Effects CRC Press, Boca Raton FL, 1991. Hermes, J. D. and Cleland, W. W. J. Am. Chem. Soc. 106, 7263 (1999))...

Klinman, J.P. (1981). Probes of mechanism and transition-state structure in the alcohol dehydrogenase reaction. CRC Crit. Rev. Biochem. 10, 39-78... 

Reaction Mechanism and Kinetic Constants for a Reaction Catalyzed by Glucose-6-Phosphate Dehydrogenase... 

The principles of enzyme catalysis discussed on p. 90 can be illustrated using the reaction mechanism of lactate dehydrogenase (LDH) as an example. 

This zinc metalloenzyme [EC 1.1.1.1 and EC 1.1.1.2] catalyzes the reversible oxidation of a broad spectrum of alcohol substrates and reduction of aldehyde substrates, usually with NAD+ as a coenzyme. The yeast and horse liver enzymes are probably the most extensively characterized oxidoreductases with respect to the reaction mechanism. Only one of two zinc ions is catalytically important, and the general mechanistic properties of the yeast and liver enzymes are similar, but not identical. Alcohol dehydrogenase can be regarded as a model enzyme system for the exploration of hydrogen kinetic isotope effects. 

A sequential enzyme-catalyzed reaction mechanism in which two substrates react to form two products and in which there is a preferred order in the binding of substrates and release of products. Several enzymes have been reported to have this type of binding mechanism, including alcohol dehydrogenase , carbamate kinase , lactate dehydrogenase , and ribitol dehydrogenase. ... 

An enzyme reaction mechanism involving A binding before B and followed with the random release of products. In the absence of products and abortive complexes, the steady-state rate expression is identical to the rate expression for the ordered Bi Bi mechanism . A random on-ordered off Bi Bi mechanism has been proposed for a mutant form of alcohol dehydrogenase. ... 

Studies of product inhibition357,358 and initial velocity359 of the dehydrogenase reaction are consistent with a hexa uni ping-pong mechanism,359 which requires that an irreversible step of the process should occur in the reaction sequence prior to addition of the second molecule of NAD to the enzyme. The irreversible step is, most probably, formation of the uronic acid (90a) from the aldehyde 93, and several mechanisms may be written to explain the kinetics ob-... 

FIG. 1. —Possible Mechanism for the Uridine 5 -(oi-D-Glucopyranosyl Pyrophosphate) Dehydrogenase Reaction. [The dashed line shows the reaction in the presence of the aldehyde 93.]... 

Describe the subunit structure of the enzyme pyruvate dehydrogenase. Discuss the functioning of each of the coenzymes that are associated with these subunits and write detailed mechanisms for each step in the pyruvate dehydrogenase reaction. 

Catalytic Reaction Mechanism of Drosophila ADH, a Short-Chain Dehydrogenase... 

Alcohol dehydrogenases are enzymes that are well known from physiological and biochemical studies on the primary metabolism of cells. Several ADHs are commercially available and for some of them such as the ADH from yeast or liver details concerning the structure and reaction mechanism have been elucidated. For preparative applications however they seldom meet the requirements and new enzymes are needed for this field. 

Enzymatic Reaction Mechanisms I Lactate dehydrogenase is one of the many enzymes that require NADH as coenzyme. It catalyzes the conversion of pyruvate to lactate ... 

Answer The mechanism is the same as that of the alcohol dehydrogenase reaction (Fig. 14-13, p. 547). 

Answer Oxidative decarboxylation involving NADP+ or NAD+ as the electron acceptor the a-ketoglutarate dehydrogenase reaction is also an oxidative decarboxylation, but its mechanism is different and involves different cofactors TPP, lipoate, FAD, NAD+, and CoA-SH. 

---