SUBSTITUTIONS IN THE ACTIVE-SITE OF CHLORAMPHENICOL ACETYLTRANSFERASE - ROLE OF A CONSERVED ASPARTATE
SUBSTITUTIONS IN THE ACTIVE-SITE OF CHLORAMPHENICOL ACETYLTRANSFERASE - ROLE OF A CONSERVED ASPARTATE
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DOI：
10.1021/bi00419a032
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发表时间：
1988-09-20
期刊：
影响因子：
2.9
通讯作者：
SHAW, WV
中科院分区：
文献类型：
--
作者：
LEWENDON, A;MURRAY, IA;SHAW, WV
用定点突变的方法研究了氯霉素乙酰转移酶（CAT）中保守的Asp-199的作用。用丙氨酸取代Asp-199会产生一种不耐热突变酶（Ala-199 CAT），其kcat降低（13倍），但Km值与野生型CAT相似。用天冬酰胺取代会产生一种热稳定突变酶（Asn-199 CAT），其kcat大大降低（1500倍）。此外，Asn-199 CAT显示与亲和试剂3-（溴乙酰基）氯霉素的异常失活动力学。这些结果有利于结构作用的Asp-199，而不是一个催化剂，在保持与晶体学证据的参与的Asp-199在一个紧密的盐桥与Arg-18。用缬氨酸取代Arg-18产生与Ala-199 CAT具有相似性质的突变酶（瓦尔-18 CAT）。His-195的催化咪唑似乎在构象上受到N1-H与同一残基的羰基氧之间的氢键以及与Tyr-25的环堆积的限制。
The role of conserved Asp-199 in chloramphenicol acetyltransferase (CAT) has been investigated by site-directed mutagenesis. Substitution of Asp-199 by alanine results in a thermolabile mutant enzyme (Ala-199 CAT) with reduced kcat (13-fold) but similar Km values to wild type CAT. Replacement by asparagine gives rise to a thermostable mutant enzyme (Asn-199 CAT) with much reduced kcat (1500-fold). Furthermore, Asn-199 CAT shows anomalous inactivation kinetics with the affinity reagent 3-(bromoacetyl)chloramphenicol. These results favor a structural role for Asp-199 rather than a catalytic one, in keeping with crystallographic evidence for involvement of Asp-199 in a tight salt bridge with Arg-18. Replacement of Arg-18 by valine results in a mutant enzyme (Val-18 CAT) with similar properties to Ala-199 CAT. The catalytic imidazole of His-195 appears to be conformationally constrained by hydrogen bonding between N1-H and the carbonyl oxygen of the same residue and by ring stacking with Tyr-25.