Martín J, Slade A, Aitken A, Arche R, Virden R
Departamento de Bioquímica y Biología Molecular I, Facultad de Química, Universidad Complutense, Madrid, Spain.
Biochem J. 1991 Dec 15;280 ( Pt 3)(Pt 3):659-62. doi: 10.1042/bj2800659.
The site of reaction of penicillin acylase from Kluyvera citrophila with the potent inhibitor phenylmethanesulphonyl fluoride was investigated by incubating the inactivated enzyme with thioacetic acid to convert the side chain of the putative active-site serine residue to that of cysteine. The protein product contained one thiol group, which was reactive towards 2,2'-dipyridyl disulphide and iodoacetic acid. Carboxymethylcysteine was identified as the N-terminal residue of the beta-subunit of the carboxy[3H]methylthiol-protein. No significant changes in tertiary structure were detected in the modified penicillin acylase using near-u.v. c.d. spectroscopy. However, the catalytic activity (kcat) with either an anilide or an ester substrate was decreased in the thiol-protein by a factor of more than 10(4). A comparison of sequences of apparently related acylases shows no other extensive regions of conserved sequence containing an invariant serine residue. The side chain of this residue is proposed as a candidate nucleophile in the formation of an acyl-enzyme during catalysis.
通过将失活的酶与硫代乙酸孵育，以将假定的活性位点丝氨酸残基的侧链转化为半胱氨酸的侧链，研究了嗜柠檬酸克吕沃尔氏菌青霉素酰化酶与强效抑制剂苯甲磺酰氟的反应位点。蛋白质产物含有一个硫醇基团，该基团对2,2'-二吡啶二硫化物和碘乙酸有反应活性。羧甲基半胱氨酸被鉴定为羧基[³H]甲基硫醇蛋白β亚基的N端残基。使用近紫外圆二色光谱法未检测到修饰后的青霉素酰化酶的三级结构有明显变化。然而，硫醇蛋白对苯胺或酯底物的催化活性（kcat）降低了10⁴倍以上。对明显相关的酰化酶序列的比较表明，没有其他包含不变丝氨酸残基的广泛保守序列区域。该残基的侧链被认为是催化过程中形成酰基酶时的候选亲核试剂。
