Enzyme-induced inactivators

The efficiency of inactivation by covalent bond formation vs release of the reactive species into solution has been described by its partition ratio. The most efficient inactivators have catalytic partition ratios of 0, in which case each inhibitor molecule leads to inactivation of the enzyme. To this date, many of these inhibitors have been designed, and alternative names like suicide substrate, Trojan Horse inactivator, enzyme induced inactivator, inhibitor, and latent inactivator have been used for this class of inhibitors. A number of comprehensive reviews are available (26—32). 

John, R. A. Enzyme-Induced Inactivation of Pyridoxal Phosphate-Dependent Enzymes Approaches to the design of specific inhibitors, in Enzyme Inhibitors as Drugs, (ed.) Sandler, M p. 73, London, Macmillan Press 1980... 

Bacteria produce chromosomady and R-plasmid (resistance factor) mediated P-lactamases. The plasmid-mediated enzymes can cross interspecific and intergeneric boundaries. This transfer of resistance via plasmid transfer between strains and even species has enhanced the problems of P-lactam antibiotic resistance. Many species previously controded by P-lactam antibiotics are now resistant. The chromosomal P-lactamases are species specific, but can be broadly classified by substrate profile, sensitivity to inhibitors, analytical isoelectric focusing, immunological studies, and molecular weight deterrnination. Individual enzymes may inactivate primarily penicillins, cephalosporins, or both, and the substrate specificity predeterrnines the antibiotic resistance of the producing strain. Some P-lactamases are produced only in the presence of the P-lactam antibiotic (inducible) and others are produced continuously (constitutive). 

When metabolic activation is used, S9 mix should not exceed 1-10 percent of the culture medium by volume. It has been shown that the S9 mix is clastogenic in CHO cells and mouse lymphoma cells (Cifone et al., 1987 Kirkland et al., 1989) but not in human lymphocytes, where blood components can inactivate active oxygen species which could cause chromosome damage. When S9 mix from animals treated with other enzyme-inducing agents such as phenobarbitone/beta-naphtho-flavone, is used, clastogenesis may be minimized (Kirkland et al., 1989). 

The NNRTIs inhibit viral reverse transcriptase by binding adjacent to its active site and inducing a conformational change that causes the enzyme s inactivation. When combined with NRTIs or protease inhibitors,... 

Studies on irradiated solutions of phenolase showed that the enzyme was inactivated. The radiation-induced changes were found to be different from those reported to occur upon denaturation of proteins. Infrared absorption spectra revealed that deamination had occurred. Acid- and basebinding groups were reduced in number rather than increased, and the optical rotation became more dextrorotatory than levorotatory. It was fur-... 

We have shown that there is good agreement between dissociation constants obtained by kinetic studies, and by measurements of the substrate-induced enzyme inactivation (4) the latter may yield true equilibrium constants, as is the case for Kj determinations, free from kinetic variables that may be present in estimations of Km (6). However, the constants determined by inducing inactivations cannot be accepted without properly evaluating other factors (14). 

Generally it is possible to activate enzymes under pressure, but at high hydrostatic pressure a decrease in activity occures due to pressure induced inactivation. The impact of pressure on the reaction rates is quite complex because of the different susceptibilities of the catalytic steps. It should be useful to perform enzyme catalysed reactions under hydrostatic pressure because pressure and temperature can have contrary effects. 

Reminiscent of the irreversible inhibition of GABA-T by ethanolamine-O-sulfate (10). which involves enzyme-induced P elimination of sulfate to generate an electrophilic Michael acceptor, P-haloamino acids have been found to lead to irreversible inhibition via 8-elimination mechanisms. Thus bacterial alanine racemase is irreversibly inhibited by P-chloro-D-alanine (24), P-fluoroalanine (25) and by P,P,P-trifluoroalanine (26). P,P,P-Trifluoroalanine has also been found to be an irreversible inactivator of y-cystathionase (26, 27). the enzyme previously shown to be inactivated by propargylglycine (7). 

In the preceding sections we have noted the biosynthesis and/or catabolism of cyclopropyl moieties by carbocation, by carbanion and by radical processes in specific contexts. At least two naturally occurring cyclopropyl metabolites and several synthetic ones have been observed to induce inactivation of specific target enzymes. The inactivation is presumptively related to chemical reactivity of loci at or adjacent to the cyclopropane equivalent. In the case of hypoglycine A (7) the methylene cyclopropane group is the problematic moiety, while in coprine (29) the hemiaminal is a latent cyclopropanone equivalent we shall analyze the proposed enzyme killing routes for each. Then we will turn to cyclopropyl... 

Kornblatt, M.J. Hoa, G.H.B. The pressure-induced inactivation of mammalian enolases is accompanied by dissociation of the dimeric enzyme. Arch. Biochem. Biophys. 

Groutas, W. C., Stanga, M. A., Brubaker, M. J. C NMR evidence for an enzyme-induced Lessen rearrangement in the mechanism-based inactivation of a-chymotrypsin by 3-benzyl-N-((methylsulfonyl)oxy)succinimide. J. Am. Chem. Soc. 1989, 111, 1931-1932. 

Heparin acts as a catalyst for antithrombin III (AT III), increasing its activity by approximately a thousand times. Antithrombin III is a plasma enzyme that inactivates certain activated serine proteases of the coagulation cascade, most importantly activated factors II (thrombin) and X. The larger heparin species (found in unfractionated heparin) catalyzes the inactivation of activated factors II and X. In contrast, LMWH chiefly inactivates activated factor X. The final effect of both is systemic anticoagulation. Heparin also possesses inherent platelet-aggregating properties and may also induce the production of platelet-aggregating antibodies. Heparin can inhibit aldosterone synthesis. 

Some of these differences can be attributed to variations in detoxication mechanisms. For example, the loss of consciousness induced in several species of laboratory animals by hexobarbital (a barbiturate derivative that depresses the central nervous system (CNS)) shows marked differences these are attributable to the activity of the detoxication enzyme that inactivates this chemical. In the mouse, the activity of the detoxifying enzyme is 16-fold greater than that in the dog, which is reflected by 12 min of hexobarbital-induced sleep in the mouse versus 315 min of sleep in the dog. There are other examples of species-related differences in the ability to detoxify chemicals that consequently result in differences in toxicity. Other examples include the industrial chemicals, ethylene glycol and aniline. Ethylene glycol is metabolized to oxalic acid, which is responsible for its toxicity, or to carbon dioxide. The rank order of ethylene glycol toxicity in animals is as follows cat rat rabbit this is the same for the extent of oxalic acid production. Aniline is metabolized in the cat and dog mainly to o-aminophenol, and these species are more prone to toxicity however, in the rat and hamster aniline is metabolized mainly to I-aminophenol and thus these species are less susceptible to aniline toxicity. 

Because of their enzyme-inducing effects, barbiturates can cause increased inactivation of other compounds (anticoagulants, phenytoin, theophylline, digoxin, glucocorticoids, etc.). This may lead to serious problems with drug interactions. 

It is also noteworthy that, in the presence of ThDP and pyruvate only, some PDHcs have been reported to undergo slow inactivation . This inactivation could be explained using a combination of now known facts. If on decarboxylation of pyruvate the enamine undergoes slow air-oxidation concomitant with turnover, the resulting 2-acetylThDP can periodically transfer its acetyl group to a CysSH on the enzyme, rather than to water. This would lead to inactivation, according to the model for fluoropyruvate-induced inactivation discussed in equation 9. 

The main conclusion from this study is that both PP and LM firefly luciferases have similar kinetic mechanisms characterized by significant inactivation of the enzyme induced by its interaction with the substrates. The difference in kinetic properties for both enzymes is mainly in the reaction rates for formation and dissociation of the luciferin-luciferase comlex. The addition of pyrophosphate to the reaction mixture increases the reaction yield due to enhanced regeneration of active enzyme from the enzyme-product complex. 

---