Dehydrogenases substrate competitive

It has been known since the early studies of Kearney (192) that succinate dehydrogenase undergoes reversible activation by substrates, competitive inhibitors, and phosphate. The activation of succinate dehydrogenase was shown to be a characteristic of both the soluble and particle-bound enzyme and a slow process requiring many minutes of incubation with the activator at ambient or higher temperatures (activation energy = 31-33 kcal/mole). It has been suggested that the enzyme exists in a free equilibrium between the unactivated and the activated forms, and that the activator interacts with the latter and establishes a new equilibrium in favor of the activated state of the enzyme (23, 25, 193 see also 194 for an expanded mechanism). 

A. Methanol Methanol (wood alcohol) is sometimes used by alcoholics when they are unable to obtain ethanol and is a constituent of windshield cleaners and canned heat Intoxication from methanol alone may include visual dysfunction, gastrointestinal distress, shortness of breath, loss of consciousness, and coma. Methanol is metabolized to formaldehyde and formic acid, which can cause severe acidosis, retinal damage, and blindness. The formation of formaldehyde is retarded by prompt intravenous administration of ethanol, which acts as a preferred substrate for alcohol dehydrogenase and competitively inhibits the oxidation of methanol (Figure 23-2). 

For the expression of ras oncogene function, Ras must bind to GTP. In the metabolic pathway of GMP synthesis, IMP is converted to XMP by IMP dehydrogenase, and the XMP is converted to GMP by GMP synthetase. Oxanosine itself does not inhibit IMP dehydrogenase, but oxanosine 5 -monophosphate inhibits IMP dehydrogenase almost competitively with the substrate. Therefore, it is likely that... 

FIGURE 14.14 Structures of succinate, the substrate of succinate dehydrogenase (SDH), and malouate, the competitive inhibitor. Fumarate (the product of SDH action on succinate) is also shown. 

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. 

Alberty analyzed the anion effect on pH-rate data. He first considered a one-substrate, one-product enzyme-catalyzed reaction in which all binding interactions were rapid equilibrium phenomena. He obtained rate expressions for effects on F ax and thereby demonstrating how an anion might alter a pH-rate profile. He also considered how anions may act as competitive inhibitors. The effect of anions on alcohol dehydrogenase has also been investigated. Chloride ions appear to affect the on- and off-rate constants for NAD and NADH binding. See also pH Studies Activation Optimum pH... 

A uridine 5 -(2-acetamido-2-deoxy-a-D-glucopyranosyl pyrophosphate) dehydrogenase has been obtained in partially purified form from extracts of a strain of Achromobacter georgiopolitanum.367 The product 90b was found to be a competitive inhibitor of the reaction when the concentration of the substrate (89b) was varied such inhibition should occur if the kinetic mechanism of the reaction is similar to that of the dehydrogenase of 89a. Substitution of 89a for 89b, or of NAD phosphate (NADP ) for NAD , is possible, but results in a 10-fold decrease of the reaction rate. 

The malate, oxaloacetate, and fumarate analogues 3-arsonolactate (86), 3-arsonopyruvate (68), and CE)-3-arsonoacrylate (87,88) have been made Ali and Dixon (88) found that the fumarate and malate analogues were not substrates for fumarate hydratase, but competitive inhibitors, arsonoacrylate, H203As—CH=CH—COOH, with fumarate ( / 1.8) and arsonolactate, H203As—CH2—CHOH—COOH, with malate (KJKm 1.6). Incidentally, although phosphonopyruvate is a poor substrate for malate dehydrogenase (89,90), 3-(hydroxyphosphinoyl)pyru-vate, HO—P(H)(0)—CH2—CO—COOH, is much better (89). It proved impossible to show the reverse reaction with arsonolactate (89, 91). 

Uncompetitive inilibitors of liver alcohol dehydrogenase (Chapter 15) could be used to treat cases of poisoning by methanol or ethylene glycol.81 83 The aim is to prevent rapid oxidation to the toxic acids HCOOH and HOCH2COOH, which lower blood pH, while the alcohols are excreted. Uncompetitive inhibitors have an advantage over competitive inhibitors as therapeutic agents in that the inhibition is not overcome when the substrate concentration is saturating.84... 

Competitive inhibitors often closely resemble in some respect the substrate whose reactions they inhibit and, because of this structural similarity, compete for the same binding site on the enzyme. The enzyme-inhibitor complex either lacks the appropriate reactive groups or is held in an unsuitable position with respect to the catalytic site of the enzyme which results in a complex which does not react (i.e. gives a dead-end complex). The inhibitor must first dissociate before the true substrate may enter the enzyme and the reaction can take place. An example is malonate, which is a competitive inhibitor of the reaction catalysed by succinate dehydrogenase. Malonate has two carboxyl groups, like the substrate, and can fill the substrate binding site on the enzyme. The subsequent reaction, however, requires that the molecule be reduced with the formation of a double bond. If malonate is the substrate, this cannot be achieved without the loss of one of the carboxy-groups and therefore no reaction occurs. 

The classic example of competitive inhibition is inhibition of succinate dehydrogenase, an enzyme, by the compound malonate. Hans Krebs first elucidated the details of the citric acid cycle by adding malonate to minced pigeon muscle tissue and observing which intermediates accumulated after incubation of the mixture with various substrates. The structure of malonate is very similar to that of succinate (see Figure 1). The enzyme will bind malonate but cannot act further on it. That is, the enzyme and inhibitor form a nonproductive complex. We call this competitive inhibition, as succinate and malonate appear to compete for the same site on the enzyme. With competitive inhibition, the percent of inhibition is a function of the ratio between inhibitor and substrate, not the absolute concentration of inhibitor. 

A classic example of competitive inhibition is the inhibition of succinate dehydrogenase by malonate, a structural analogue of succinate. Competitive inhibitors are usually structural analogues of the substrate, the molecule with which they are competing. They bind to the active site but either do not have a structure that is conducive to enzymatic modification or do not induce the proper orientation of catalytic amino acyl residues required to affect catalysis. Consequently, they displace the substrate from the active site and thereby depress the velocity of the reaction. Increasing [S] will displace the inhibitor. 

Many substances interact with enzymes to lower their activity that is, to inhibit them. Valuable information about the mechanism of action of the inhibitor can frequently be obtained through a kinetic analysis of its effects. To illustrate, let us consider a case of competitive inhibition, in which an inhibitor molecule, I, combines only with the free enzyme, E, but cannot combine with the enzyme to which the substrate is attached, ES. Such a competitive inhibitor often has a chemical structure similar to the substrate, but is not acted on by the enzyme. For example, malonate (-OOCCH2COO-) is a competitive inhibitor of succinate (-OOCCH2CH2COO-) dehydrogenase. If we use the same approach that was used in deriving the Michaelis-Menten equation together with the additional equilibrium that defines a new constant, an inhibitor constant, A),... 

A competitive inhibitor of an enzyme will typically structurally resemble a substrate of the enzyme. Thus malonate (methanedicarboxylate OOC-CH2-COO ) is structurally similar to succinate (ethanedicarboxylate OOC CH2 CH2 COO ) and is a competitive inhibitor of the oxidoreductase succinate dehydrogenase that catalyses the reaction ... 

It has been known since the work of Hopkins and his colleagues in 1938 (186) that succinate dehydrogenase contains —SH groups essential for the catalytic activity of the enzyme, and that substrates and competitive inhibitors protect the enzyme against inhibition by —SH reagents. 

Examples of Rapid Reversible Inhibitors. Competitive inhibitors are often similar in structure to one of the substrates of the reaction they are inhibiting. Inhibitors of this type are sometimes called substrate analogs and their binding affinity (K ) usually approximates that of the substrate. One of the first reactions inhibited by a substrate analog was that catalyzed by succinate dehydrogenase (Equation 17.24). 

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