Tobin M B, Cole S C, Kovacevic S, Miller J R, Baldwin J E, Sutherland J D
Dyson Perrins Laboratory, Oxford, UK.
FEMS Microbiol Lett. 1994 Aug 1;121(1):39-46. doi: 10.1111/j.1574-6968.1994.tb07073.x.
Using a high level Escherichia coli expression system for the Penicillium chrysogenum penDE gene, we have produced acyl-coenzyme A: isopenicillin N acyltransferase (AT) enzymes containing amino acid substitutions at three conserved Ser residues. Chosen for study based on amino acid sequence homologies to other proteins, Ser227, Ser230 and Ser309 were changed to Cys or Ala to assess amino acid side chain involvement in proenzyme cleavage and AT enzyme mechanism. Substitutions at Ser230 had no effect on proenzyme cleavage, acyl-coenzyme A: IPN acyltransferase (IAT) or acyl-coenzyme A:6-aminopenicillanic acid acyltransferase (AAT) activities. While Ser227-->Cys had no effect, Ser227-->Ala produced uncleaved proenzyme lacking both AAT and IAT activities, suggesting that the presence of a nucleophilic side chain at this residue is required for proenzyme cleavage and AT activity. Substitution of Ser309-->Cys did not appreciably prevent proenzyme cleavage, IAT or AAT activity. Recombinant AT (recAT) proenzyme containing Ser309-->Ala was cleaved; however, IAT and AAT activities were not observed. This separation of proenzyme cleavage from IAT and AAT activities has not been previously observed, and suggests that Ser309 is involved in substrate acylation.
利用用于产黄青霉penDE基因的高水平大肠杆菌表达系统，我们制备了在三个保守丝氨酸残基处含有氨基酸取代的酰基辅酶A：异青霉素N酰基转移酶（AT）。基于与其他蛋白质的氨基酸序列同源性选择进行研究，将Ser227、Ser230和Ser309替换为半胱氨酸或丙氨酸，以评估氨基酸侧链在酶原裂解和AT酶机制中的作用。Ser230的取代对酶原裂解、酰基辅酶A：IPN酰基转移酶（IAT）或酰基辅酶A：6-氨基青霉烷酸酰基转移酶（AAT）活性没有影响。虽然Ser227→Cys没有影响，但Ser227→Ala产生了未裂解的酶原，缺乏AAT和IAT活性，这表明该残基处亲核侧链的存在是酶原裂解和AT活性所必需的。Ser309→Cys的取代并未明显阻止酶原裂解、IAT或AAT活性。含有Ser309→Ala的重组AT（recAT）酶原被裂解；然而，未观察到IAT和AAT活性。酶原裂解与IAT和AAT活性的这种分离以前未被观察到，这表明Ser309参与底物酰化。
