In this work, the inactivation at pH 7.0 of liver alcohol dehydrogenase by iodoacetamide and a series of six haloacids has been studied, and the kinetic constants determined. Enzyme inactivation was compared with the model alkylation of a metal-thiol and a thiolate anion free in solution. The following conclusions resulted.1. Inactivation of liver alcohol dehydrogenase by iodoacetamide is a direct thiol alkylation, while inactivation by selective alkylation of Cys-46 by the haloacids is facilitated by reversible complex formation.2. Inactivation half-time for the haloacids ranged over 4-190 min, a difference mainly caused by dissimilar chemical reactivities rather than diverse fitting in the active site.3. The thiol of Cys-46 is alkylated as a zinc-thiol complex. It is, as such, not especially reactive; indeed it has a nucleophilic reactivity similar to that observed with the model compound free in solution.4. Affinity labelling of liver alcohol dehydrogenase by haloacids compared with alkylation of the similar group free in solution illustrates enzymatic catalysis by reversible complex formation. With the present series of 'substrates' a rate enhancement of up to 58000 is seen.The a-haloacids iodoacetate, bromoacetate, bromoimidazolyl propionate and chloro-imidazolyl propionate are known to alkylate cysteine-46 selectively in liver alcohol dehydrogenase [I -41. X-ray analysis has shown that Cys-46 is one of the three protein ligands to the active-site zinc ,atom [5]. Kinetic investigations have shown that the alkylalion of this residue results from the formation of a reversible complex prior to irreversible alkylation [I]. The reversible complex is formed by carboxylate binding to the general anion-binding site, identified as arginine-47 [5]. This site is part of the coenzyme-binding site, and is where the pyrophosphate group of the coenzyme binds. Several anions thus bind in competition with both coenzyme and a-haloacid, and recently it was shown that even phosphate anions bind to this site [6]. Selective alkylation of Cys-46 with iodoacetate has been taken as a typical example of affinity labelling or active-site-directed 'irreversible inhibition [7].So far, attempts to affinity label liver alcohol dehydrogenase with other haloacids have been unsuccessful, even though slow inactivation has been reported with 2-iodopropionate, 3-bromopropionate, 3-iodopropionate and also with the 4, 5 and 6 carbon bromoacids [8,9]. Interpretation of these inactivation results has suffered from a lack of knowledge of the chemical reactivity of the different haloacids. In a recent study, the alkylating reactivity of a series of alkyl halides was measured by reaction with the free thiolate anion ofcysteine [lo]. The chemical reactivity of the haloacids varied greatly. To understand affinity labelling of liver alcohol clehydrogenase with haloacids, it is therefore of vital importance to compare the enzyme reaction with a model reaction involving the corresponding group free in solution.In this work, iodoacetamide and a series of h...
The effect of imidazole on the inactivation of liver alcohol dehydrogenase by alkylation of Cys-46 with iodoacetate, bromoacetate, 2-bromopropionate, 3-bromopropionate, 2-bromobutyrate and iodoacetamide has been studied at pH 7.0. Imidazole promoted inactivation with all the compounds but 2-bromobutyrate.Enzyme inactivation with haloacids was faster in a ternary enzyme-imidazole-haloacid complex comparcd to a binary enzyme-haloacid complex. Inactivation with iodoacetamide, which is a direct bimolecular reaction, was faster with the binary enzyme-imidazole complex as compared to the free enzyme. Results of haloacid and iodoacetamide inactivation in the presence of imidazole were fitted, by nonlinear regression analysis, to the rate expressions for the proposed mechanisms and the kinetic parameters resulted. Imidazole was also found to promote inactivation of the cobalt-substituted and cadmium-substituted liver alcohol dehydrogenases.Cys-46 is alkylated as a metal-thiol complex. Imidazole, when binding to the active-site metal, donates 0-electrons to the metal atom, which distributes the increased electron density further to the other ligands. The increased nucleophilicity of the sulphur of Cys-46 results in promoted alkylation. Proof that the imidazole promotion effect is caused by a displaced electron distribution in the active-site coordination unit is provided by imidazole also promoting the alkylation of the model thiol, zinc-mercaptoethanol.The unsaturated nitrogen heterocycle imidazole is an unidentate metal ligand. It binds to liver alcohol dehydrogenase forming a binary complex, or in the presence of coenzyme a ternary complex [l]. Direct coordination of one of the nitrogen atoms to the active-site metal has been proved by crystallographic studies on the enzyme-imidazole complex [2] and by perturbation of the cobalt enzyme spectrum upon addition of imidazole [3,4].The mechanism of inactivation of liver alcohol dehydrogenase with iodoacetate has been established from kinetic studies of the inactivation reaction [5]. It is a typical example of an affinity labelling mechanism. A complex is formed by reversible binding to the general anion-binding site, prior to the irreversible alkylation of Cys-46, one of the three protein ligands to the active-site zinc atom. Catalysis by the affinity labelling mechanism has recently been shown to operate not only with haloacetates but also for 2-bromopropionate, 3-bromopropionate, 2-bromobutyrate and 2-bromo-3-(5-imidazoy1)propionate [6]. Iodoacetamide on the other hand, inactivates the enzyme by direct alkylation.Before the mechanism of inactivation was established, Evand and Rabin [7] observed that imidazole promoted the alkylation of horse liver alcohol dehydrogenase with both iodoacetate and iodoacetamide, and that the rate of alkylation with iodoacetate became pH-independent. This stimulation or rate enhancement of alkylation has since been referred to as thc 'promotion cffect', and several workers have studied it [8,9]. Imidazole promotion has been shown to improve ...
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