丁香假单胞菌乙烯形成酶组氨酸残基的定点突变

Kazuhiro Nagahama , Kuniaki Yoshino , Masayoshi Matsuoka , Sumio Tanase , Takahira Ogawa , Hideo Fukuda
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引用次数: 13

摘要

采用谷氨酰胺取代10个组氨酸残基的定点诱变方法,研究了组氨酸残基在丁香假单胞菌乙烯形成酶(EFE)催化2-氧戊二酸转化为乙烯中的作用。在大肠杆菌中表达的突变酶被纯化至均匀性,并进行了体外Km、kcat和热稳定性测试。H305Q和H335Q两个突变efe的相对kcat分别为40%和60%。然而,His-189或His-233的突变导致活性完全丧失,这意味着这些残基在铁的结合中起着重要作用。其他突变型酶的kcat值比野生型酶低11 ~ 55倍。对于6个部分失活的突变EFEs(但不包括H305Q和H335Q),在30°C下热失活的一级速率常数比野生型酶高11- 24倍。值得注意的是,H268Q的热失活一阶速率常数与H335Q相同。H268的替代导致kcat值急剧下降(相对kcat值为1.8%)。这表明His-268的取代可能导致EFE活性位点的破坏。对纯化的突变体酶进行热失活研究发现,一些突变体酶,如H168Q和H116Q,比野生型酶更耐热。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Site-directed mutagenesis of histidine residues in the ethylene-forming enzyme from Pseudomonas syringae

The roles of histidine residues in the catalysis of the transformation of 2-oxoglutarate into ethylene via the ethylene-forming enzyme (EFE) from Pseudomonas syringae were studied using site-directed mutagenesis with substitution of glutamine for ten individual histidine residues. The mutant enzymes, which were expressed in Escherichia coli, were purified to homogeneity, and assayed in vitro for Km, kcat and thermostability. The relative kcat of two mutated EFEs, H305Q and H335Q, were 40% and 60%, respectively. However, a mutation at either His-189 or His-233 caused a total loss of activity, implying that these residues play important roles in the binding of iron. The kcat values for other mutant enzymes were 11-to 55-fold less than that for the wild-type enzyme. For six partially inactive mutated EFEs (but not for H305Q or H335Q), the first order rate constants for heat inactivation at 30°C were 11-to 24-fold higher than for the wild-type enzyme. It is noteworthy that the value of the first order rate constant for heat inactivation of H268Q was identical to that of H335Q. The substitution of H268 resulted in a drastic decrease of the kcat value (relative kcat was 1.8%). This suggests that the substitution at His-268 may cause the disruption of the active site of the EFE. Heat inactivation studies with the puridied mutant enzymes revealed that some mutant enzymes, such as H168Q and H116Q, were more thermolabile than the wild-type enzyme.

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