Partial inactivation of cytochrome c oxidase by nonpolar mercurial reagents

Purified beef heart cytochrome c oxidase is inactivated to the extent of 35 to 50% by the nonpolar mercurial reagents mercuric chloride and ethylmercuric chloride. The inactivation is complete within 5 min. In titrations of activity, the plateau level of inactivation is attained at added ethylmercuric chloride:heme a ratios of about 1:1. Up to 3 mercury atoms/heme a are bound to the oxidase, although only the first of these affects its enzymatic activity. Incubation of the ethylmercury-modified oxidase with sulfhydryl compounds reverses the inactivation, with 2,3-dimercaptopropanol being most effective of the reagents tested. Spectrophotometric and polarographic assays of enzymatic activity show that Km values for the native and the ethylmercury-modified enzymes are practically indistinguishable, and that the partial inactivation observed for the latter is reflected exclusively in a lower value of Vmax compared to that of the native enzyme. Based on these results, we propose that ethylmercuric chloride reacts with a single crucial--SH group per heme a, and that electron transfer processes in the modified product are partially inhibited.     

Beta-lactamase inactivation by mechanism-based reagents

The mechanistic pathway followed by the E. coli RTEM beta-lactamase has been studied with a view to clarifying the mode of action of a number of recently discovered inactivators of the enzyme. There is clear evidence that the beta-lactamase-catalysed hydrolysis of the 7-alpha-methoxycephem, cefoxitin, proceeds via an acyl-enzyme intermediate. An analysis of the inactivation reactions of all the known beta-lactam derivatives that result in irreversible loss of enzyme activity permits the identification of three structural features required for a beta-lactamase inactivator. The application of these principles suggests a new group of mechanism-based inactivators of the enzyme: the sulphones of N-acyl derivatives of 6-beta-aminopenicillanic acid that are themselves poor substrates for the enzyme. These sulphones are powerful inactivators of the beta-lactamase.     

Torpedo acetylcholinesterase is inactivated by thiol reagents

A number of sulphydryl reagents inhibit AChE of Torpedo california with pseudo-first-order kinetics, and inhibition can be retarded by quaternary ligands which bind at either the catalytic or peripheral anionic binding sites. Colorimetric determination with one of the inhibitory sulphydryl agents, 5,5'-dithiobis (2-nitrobenzoic acid), reveals the presence of a single thiol group per catalytic subunit; our data thus suggest that inhibition is achieved by reaction with the single free sulphydryl group of Cys231.     

Xanthine oxidase converts nitric oxide to nitroxyl that inactivates the enzyme

Xanthine oxidase (XO) was found to convert nitric oxide (NO* ) released from spermine-NONOate to nitroxyl (HNO), the one-electron reduction product of NO*, in the presence of its substrate hypoxanthine under anaerobic conditions. Under these conditions, XO lost its activity. Upon aerobic incubation of XO with its substrate, neither conversion of NO* to HNO nor inactivation of the enzyme was observed. Angeli's salt (an HNO generator) or synthetic peroxynitrite inactivated XO at low concentrations, whereas high concentrations of diethylamine-NONOate (an NO* donor) and SIN-1 (which generates peroxynitrite by releasing both NO* and superoxide) were required to inactivate XO. These results suggest that HNO generated by XO under anaerobic conditions inactivates XO. As both XO and NO* synthase are activated and/or induced in ischemia-reperfusion injury, HNO formed by XO may contribute to pathogenesis by exerting its potent oxidation activity against a variety of biological compounds.     

Kinetics of inactivation of creatine kinase during modification of its thiol groups

Kinetics of inactivation and modification of the reactive thiol groups of creatine kinase by 5,5'-dithiobis(2-nitrobenzoic acid) or iodoacetamide have been compared, the former by following the substrate reaction in presence of the inactivator [Wang, Z.-X., & Tsou, C.-L. (1987) J. Theor. Biol. 127, 253]. The microscopic constants for the reaction of the inactivators with the free enzyme and with the enzyme-substrate complexes were determined. From the results obtained it appears that with respect to ATP both inactivators are noncompetitive whereas for creatine iodoacetamide is competitive but DTNB is not. The formation of the ternary complex protects against the inactivation by both DTNB and iodoacetamide. The inactivation kinetics is monophasic with both inactivators, but under similar conditions, the modification reactions in the presence of the transition-state analogue of creatine-ADP-Mg2+-nitrate show biphasic kinetics as also reported by Price and Hunter [Price, N.C., & Hunter, M.G. (1976) Biochim. Biophys. Acta 445, 364]. If the reactive ternary complex and the enzyme complexed with the transition-state analogue react in the same way with these reagents, the modification of one fast-reacting thiol group for each enzyme molecule leads to complete inactivation, indicating that the enzyme has to be in the dimeric state to be active.     

Contact inhibition within hemoglobin S polymer by thiol reagents

N-Ethylmaleimide, a thiol reagent, increases the solubility of deoxyhemoglobin S. We investigated which of the two reacted beta 93 cysteine residues of the Hb tetramer was responsible for the inhibition of Hb S polymerization. Accordingly we compared the solubility of equal mixtures of HbA + HbS, HbA NEM + HbS and HbA + HbS NEM. Upon deoxygenation these mixtures contain about 50% a stable and asymmetrical hybrid alpha 2A beta A beta S, alpha 2A beta A,NEM beta S or alpha 2A beta A beta S,NEM respectively and 25% parental molecules as confirmed by ion-exchange HPLC performed in anaerobic conditions. Within the hybrid molecule, beta A or beta A,NEM chain has to be present in the alpha beta dimer located in trans to the dimer which contains the only beta 6 valine residue participating in intermolecular contacts (dimer in cis), while beta S or beta S,NEM must be in cis position in the hybrid molecule. The solubility of mixtures increases 4% for HbA NEM + HbS and 20% for HbA + HbS NEM mixtures compared to HbA + HbS mixture, indicating that the inhibitory effect of N-ethylmaleimide is more effective in cis than in trans position. The absence of a major role played by N-ethylmaleimide located in trans was supported by the solubility study of a mixture of HbS + Hb Créteil beta 89 Ser----Asn. The beta 89 residue in trans next to the cysteine beta 93 modified the T structure similarly to N-ethylmaleimide, and did not affect intermolecular contacts. Crystallographic studies of molecular contacts within deoxyHbS crystals suggest that the cis inhibitory effect of N-ethylmaleimide can be explained by direct inhibition of 'external' contacts between double strands involving the CD corner of the alpha chains.     

Xanthine oxidase and endothelium dependent relaxation

Superoxide anion (O2-) generated from xanthine oxidase/xanthine has been used to decrease the half life of endothelium derived relaxing factor (EDRF). However, by itself, xanthine oxidase causes endothelium dependent relaxation. This relaxation is unrelated to the oxidative property of the enzyme since it is not inhibited by allopurinol. In addition, the relaxation is not inhibited by the cyclooxygenase inhibitor, indomethacin, or the phospholipase A2 inhibitor, p-bromophenacyl bromide. On the other hand the relaxation is inhibited by the trypsin inhibitor (TI) from chicken egg white. A similar endothelium dependent relaxation elicited by pancreatin and trypsin is also inhibited by TI. Pancreatin used in the preparation of xanthine oxidase contains trypsin, chymotrypsin and carboxypeptidase. When compared to trypsin both chymotrypsin and carboxypeptidase elicit little relaxation. Thus the endothelium dependent relaxation elicited by xanthine oxidase is likely due to contamination with trypsin. Our results emphasize that when the superoxide generating system, xanthine oxidase/xanthine is used to study the effect of oxygen radicals on EDRF, it is advantageous to ensure that only purified preparations of xanthine oxidase are used.     

Utilization of the inactivation rate of coenzyme A transferase by thiol reagents to determine properties of the enzyme-CoA intermediate.

The rate of inactivation of succinyl-CoA:3-ketoacid coenzyme A transferase by thiol reagents is increased 3 to 100 times by very low concentrations of acyl-CoA substrates. The same maximum inactivation rate is found with acetoacetyl-CoA and succinyl-CoA. The enhanced rate of inactivation is caused by the stoichiometric formation of the enzyme-CoA intermediate and an accompanying conformation change of the enzyme. The inactivation rate provides a simple assay for the amount of enzyme present as the enzyme-CoA intermediate, using only catalytic concentrations of enzyme. This technique has been utilized to measure (a) a rate constant for hydrolysis of the enzyme-CoA intermediate of 0.10 min-1 at pH 8.1; (b) a stoichiometry of two active sites per enzyme molecule; and (c) the equilibrium constants for formation of the enzyme-CoA intermediate from dilute solutions of substrates (and hence for the overall reaction) by determining the ratio of [enzyme-CoA]/[enzyme] in the presence of a series of substrate "buffers" at different ratios of [RCOO-]/[RCOSCoA]. As the total concentration of acyl-CoA and carbosylate substrates is increased, the inactivation rate is decreased. This indicates that the Michaelis complexes are protected against inactivation.     

Xanthine oxidase activity in regenerating liver

The xanthine oxidase activity of mouse regenerating liver has been shown to be elevated during the period of rapid liver growth and proliferation. This increase is evident when the enzyme activity is expressed per unit wet tissue weight, per unit nitrogen, or per cell. The adrenal cortex probably plays only a minor role in implementing this phenomenon. Further augmentation of the xanthine oxidase level of regenerating liver is not induced by the administration of large quantities of the substrate, xanthine, to the animal.     

Mitochondrially targeted antioxidants and thiol reagents

Mitochondrial oxidative damage and dysfunction contributes to a number of cell pathologies. To investigate how this damage affects cell function we have developed mitochondrially targeted antioxidants and thiol reagents by covalently linking them to lipophilic cations. The cation drives the selective accumulation of these reagents into mitochondria within cells where the antioxidants decrease oxidative damage and the thiol reagents enable measurement of the redox status of thiol proteins. In conjunction with cell and animal models of apoptosis, oxidative damage, and nitric oxide signaling, these molecules may provide new insights into the roles of mitochondria in human pathologies.