Dehydrogenases substrate inhibition

Many examples of product inhibition are to found. Some dehydrogenases are inhibited by NADH (a co-product of the reaction), e.g. PDH and isocitrate dehydrogenase (ICD), which are involved with the glycolysis and the TCA cycle are two such examples. Hexokinase isoenzymes in muscle (but not liver) and citrate synthase are inhibited by their products, glucose-6-phosphate and citrate respectively offering a very immediate fine tuning of reaction rate to match cellular requirements and possibly allowing their substrates to be used in alternative pathways. 

Other NADH dehydrogenases include NADH dehydrogenase (quinone) [EC 1.6.99.5] which catalyzes the reaction of NADH with an acceptor to produce NAD+ and the reduced acceptor. Menaquinone can serve as the acceptor substrate. This dehydrogenase is inhibited by AMP and 2,4-dinitrophenol but not by dicoumarol or folic acid derivatives. 

ISOTOPE EXCHANGE AT EQUILIBRIUM ISOTOPE TRAPPING LACTATE DEHYDROGENASE LIGAND EXCLUSION MODEL NONPRODUCTIVE COMPLEXES PRODUCT INHIBITION SUBSTRATE INHIBITION... 

ISOTOPE TRAPPING STICKY SUBSTRATES Substrate-induced conformational change, INDUCED FIT MODEL SUBSTRATE INHIBITION ABORTIVE COMPLEX FORMATION LACTATE DEHYDROGENASE LEE-WILSON EQUATION... 

At very high substrate concentrations deviations from the classical Michaelis-Menten rate law are observed. In this situation, the initial rate of a reaction increases with increasing substrate concentration until a limit is reached, after which the rate declines with increasing concentration. Substrate inhibition can cause such deviations when two molecules of substrate bind immediately, giving a catalytically inactive form. For example, with succinate dehydrogenase at very high concentrations of the succinate substrate, it is possible for two molecules of substrate to bind to the active site and this results in non-functional complexes. Equation S.19 gives one form of modification of the Michaelis-Menten equation. 

K. M. Moreton, and J. J. Holbrook, Removal of substrate inhibition in a lactate dehydrogenase from human muscle by a single residue, FEBS Lett. 1996, 399, 193-197. 

Primary alcohols are oxidized to aldehydes, n-butanol being the substrate oxidized at the highest rate. Although secondary alcohols are oxidized to ketones, the rate is less than for primary alcohols, and tertiary alcohols are not readily oxidized. Alcohol dehydrogenase is inhibited by a number of heterocyclic compounds such as pyrazole, imidazole, and their derivatives. 

The scheme in Fig. 9 suggests that lipoamide dehydrogenase functions by a simple binary complex mechanism, and this conforms to a vast body of kinetic evidence (Section, 111 0). Since the oxidized enzyme can form a complex (or complexes) with NAD and since EH2 may form stable complexes with both NAD and NADH, a classic binary complex mechanism is too simple. Indeed, the substrate inhibition patterns are very complex and do not conform to any classic pattern. Until spectral properties and rates of formation and breakdown can be measured for each of these complexes the simple binary complex mechanism must serve. 

Roberts, P., Basran, J., Wilson, E. K., Hille, R., and Scrutton, N. S., 1999, Redox cycles in trimethylamine dehydrogenase and mechanism of substrate inhibition, Biochemistry 38 14927nl4940. 

VlO. Vesell, E. S., pH dependence of lactate dehydrogenase isoenzyme inhibition by substrate. Nature 210, 421—422 (1966). 

Ftorafur) fluorouracil by thymidine/uridine phosphorylase in the liver Uracil Substrate for dihydropyridimidine dehydrogenase (DPD) inhibits degradation of fluorouracil rash, and neurotoxicity (dizziness, confusion, ataxia) ... 

Figure 9.8. The malate dehydrogenase dimer, indicating the location of the active sites in each protein plus the dimer interface. Malate dehydrogenase demonstrates substrate inhibition that has been attributed to subunit interactions and allosteric regulation by citrate, although the crystal structure of the protein reveals the absence of a separate allosteric site for citrate. See color insert.

Benzaldehyde can be produced from benzoyl formate with whole cells of Pseudomonas putida ATCC 12633 as biocatalyst119 201 (Fig. 16.6-5). Alternatively, but less effectively, mandelic acid can be used as starting material. A pH of 5.4 was found to be optimal for benzaldehyde accumulation. At this proton concentration, partial inactivation of the benzaldehyde dehydrogenase isoenzymes and activation of the benzoyl formate decarboxylase are reported. Fed-batch cultivation prevented substrate inhibition. In situ product removal is necessary to prevent product inhibition. 

Side note Methanol Poisoning. An interesting and imponant example competitive substrate inhibition is the enzyme alcohol dehydrogenase (AC in the presence of ethanol and methanol. If a person ingests methanol, Al will convert it to formaldehyde and then formate, which causes blindne Consequently, the treatment involves intravenously injecting ethanol (wh is metabolized at a slower rate than methanol at a controlled rate to tie ADH to slow the metabolism of methanol-to-formaldehyde-to-formate so l the kidneys have time to filter out the methanol which is then excreted in urine. With this treatment, blindness is avoided. For more on the met nol/ethanol competitive inhibition, see Problem P7 2Sc. 

The formation of abortive complexes of the type EAQ and EPB, a common cause of substrate inhibition of dehydrogenases (Section II, F), can also be detected in isotope exchange experiments by inhibition of all exchanges when the concentrations of A and Q or P and B are increased together. The complexes E NADH malate (34) and E NAD(P)H glutamate (44), for example, were detected in this way, and are probably responsible for the substrate inhibition observed in initial rate studies of these enzymes (Section II,F,1). The latter complex, but not the former. 

Substrate inhibition of bovine liver glutamate dehydrogenase has not been studied in such detail. It is most marked when the NAD(P) concentration is also large (11), and is relieved by ADP. This appears to be the reason why ADP activates the enzyme when large glutamate and... 

When Rudolph and Fromm used thionicotinamide adenine dinucleotide (thio-DPN) as an alternate substrate for NAD+ and varied the concentration of ethanol with liver alcohol dehydrogenase [following the reaction at 342 nm, the isosbes-tic point for thio-DPN and reduced thio-DPN (thio-DPNH)], they saw what appeared to be concave upward reciprocal plots with partial substrate inhibition in the presence of thio-DPN (38). However, the asymptote intercepts appeared to decrease with increased thio-DPN concentration, which is not what the above equations predict for a case where a minimum is present in the curve. There must have been other interactions that caused the substrate inhibition by ethanol in the presence of thio-DPN. 

This method was also used with alanine dehydrogenase with NADH as a product inhibitor and substrate inhibition levels of L-alanine (42). At first glance the observed j8 value of 4.9 suggests that a dead-end E-NADH-alanine complex caused the substrate inhibition in an ordered mechanism, however, )8 could not exceed 1.0 unless alanine had as great or greater affinity for E-NADH than its apparent Michaelis constant at low NAD, and, with NAD" present at its (as was the case), the value of j8 could not have exceeded 0.6. The equation for... 

Fig. 16 (A) Ru(bpy)3 based ECL reaction mechanism for detection NAD+-dehydrogenase substrates and (B) Ru(bpy)3 based ECL reaction ECL reaction mechanism for detection of oxidase substrates through ECL Inhibition. Reprinted with permission from Ref 17. Copyright (2011) Elsevier.

However, this case is not a common one. A very rare example is the reaction catalyzed by isocitrate dehydrogenase. In this reaction a-ketoglutarate shows a strong uncompetitive substrate inhibition versus NADPH or CO2, which does not result from combination with the enzyme-NADP complex. Presumably, it occurs by imine formation with a lysine in the central complex. The closure of the active site exposes this lysine, which must have a low enough ipKa to form an imine at neutral pH. In support of this model, oxalyl-glycine, a mimic of a-ketoglutarate that binds with equal affinity, does not show the effect (Grissom Cleland, 1988). 

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