The Reactivity of Sulfhydryl Groups at the Active Site of an R-Factor -Specified Variant of Chloramphenicol Acetyltransferase

Abstract

1. The commonly observed type I variant of chloramphenicol acetyltransferase specified by R factors is a tetrameric enzyme and contains four half-cysteine residues per identical subunit. All four thiols are reactive when the protein has been reduced in the presence of 6 M guanidine-HCl. 2. Iodoacetamide is an effective inhibitor of native chloramphenicol acetyltransferase. The incubation of iodo[14C]acetamide with enzyme leads to amido-carboxymethylation. Chloramphenicol is effective in preventing both inactivation and chemical modification, whereas acetyl-CoA fails to protect against either effect. Evidence is presented in support of the preferential reactivity of a single cysteine residue (S1) towards iodoacetamide. Iodoacetate is not an effective inhibitor and fails to react with the native enzyme under conditions which permit inactivation and chemical modification by iodoacetamide. 3. The chromogenic disulfides 2,2′-dithio-bis(pyridine) and 4,4′-dithio-bis(pyridine) are more effective at low concentrations than iodoacetamide as inhibitors of chloramphenicol acetyltransferase. Inhibition and covalent modification of chloramphenicol acetyltransferase by 2,2′-dithio-bis(pyridine) can be prevented by chloramphenicol but not by acetyl-CoA. The same half-cysteine-containing sequence (S1), adduced to be the site of alkylation by iodoacetamide, appears to be uniquely susceptible to mixed disulfide formation with 2,2′-dithio-bis(pyridine). Cyanylation of the 2-thiopyridyl-inhibited enzyme does not lead to reactivation. 4. Chloramphenicol acetyltransferase is also susceptible to inhibition by N-ethylmaleimide, p-mercuribenzoate, and 1-fluoro-2,4-dinitrobenzene, and protection is afforded in each case by chloramphenicol. Dinitrophenylation of one or more cysteine residues is inferred by the reactivation observed by incubation of the inactivated enzyme with reduced thiols. 5. The reaction of iodo[14C]acetamide with native chloramphenicol acetyltransferase followed by proteolytic digestion yields radioactive peptides corresponding to only three of the four known half-cysteine-containing sequences. The results of competition and protection experiments suggest that the S1 sequence is the preferential site of attack by iodoacetamide or 2,2′-dithio-bis(pyridine) and that it is sufficiently close to the chloramphenicol binding site to be protected by that substrate. A thiol-containing sequence designated S3 has been judged to be implicated in the binding of acetyl-CoA on the basis of (a) protection against iodoacetamide labelling and inactivation by that substrate, and (b) probable covalent and selective modification of the S3 thiol by bromoacetyl-CoA. 6. Low but reproducible rates of enzyme inactivation occur when chloramphenicol acetyltransferase is incubated in the presence of a number of CoA-related compounds. The inactivation process is not related to the low rates of hydrolysis of acyl-CoA derivatives, which have been noted in the absence of chloramphenicol. There is no evidence to suggest a sequential (‘ping-pong’) reaction mechanism with the formation of a covalent enzyme-substrate intermediate.