Fitton J E, Shaw W V
Biochem J. 1979 Feb 1;177(2):575-82. doi: 10.1042/bj1770575.
Four electrophoretic variants of chloramphenicol acetyltransferase (types A, B, C and D) found in chloramphenicol-resistant staphylococci were purified by affinity chromatography. Michaelis constants and the kinetics of inactivation with a variety of reagents for the four variants are virtually identical. Their similar amino acid compositions and near identical N-terminal sequences suggest a high degree of overall sequence homology. The thiol-specific reagents 5,5'-dithiobis-(2-nitrobenzoic acid), 2-nitro-5-thiocyanobenzoic acid and 2,2'-dithiopyridine are without significant effect on enzyme activity, whereas 1-fluoro-2,4-dinitrobenzene, N-ethylmaleimide, p-chloromercuribenzoic acid, iodoacetamide, and, particularly, bromoacetyl-CoA and diethyl pyrocarbonate are potent inhibitors. Iodoacetate is not an inhibitor. The results of chemical modification studies on the four enzyme variants and the identification of 3-carboxymethylhistidine in acid hydrolysates of one variant (type C) after inactivation with iodoacetamide suggest that a unique histidine residue may be involved in the mechanism of catalysis.
通过亲和层析法纯化了在耐氯霉素葡萄球菌中发现的四种氯霉素乙酰转移酶电泳变体（A、B、C和D型）。这四种变体的米氏常数以及用多种试剂进行失活反应的动力学实际上是相同的。它们相似的氨基酸组成和几乎相同的N端序列表明其总体序列具有高度同源性。硫醇特异性试剂5,5'-二硫代双（2-硝基苯甲酸）、2-硝基-5-硫氰基苯甲酸和2,2'-二硫代吡啶对酶活性没有显著影响，而1-氟-2,4-二硝基苯、N-乙基马来酰亚胺、对氯汞苯甲酸、碘乙酰胺，特别是溴乙酰辅酶A和焦碳酸二乙酯是有效的抑制剂。碘乙酸不是抑制剂。对这四种酶变体进行化学修饰研究的结果，以及在用碘乙酰胺失活后在一种变体（C型）的酸水解产物中鉴定出3-羧甲基组氨酸，表明一个独特的组氨酸残基可能参与催化机制。
