羟甲基胆色素原合酶中假定活性位点赖氨酸残基的研究。制备并表征了以下突变体：(a) Lys-55被谷氨酰胺取代；(b) Lys-59被谷氨酰胺取代；(c) Lys-55和Lys-59均被谷氨酰胺取代。
Investigation of putative active-site lysine residues in hydroxymethylbilane synthase. Preparation and characterization of mutants in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 have been replaced by glutamine.
作者信息
Hädener A, Alefounder P R, Hart G J, Abell C, Battersby A R
机构信息
University of Cambridge Chemical Laboratory, U.K.
出版信息
Biochem J. 1990 Oct 15;271(2):487-91. doi: 10.1042/bj2710487.
Abstract
A new construct carrying the hemC gene was transformed into Escherichia coli, resulting in approx. 1000-fold over-expression of hydroxymethylbilane synthase (HMBS). This construct was used to generate HMBS in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 were replaced by glutamine (K55Q, K59Q and K55Q-K59Q respectively). All three modified enzymes are chromatographically separable from wild-type enzyme. Kinetic studies showed that the substitution K55Q has little effect whereas K59Q causes a 25-fold decrease in Kapp. cat./Kapp. m. Treatment of K55Q, K59Q and K55Q-K59Q separately with pyridoxal 5'-phosphate and NaBH4 resulted in incomplete and non-specific reaction with the remaining lysine residues. Pyridoxal modification of Lys-59 in the K55Q mutant caused greater enzymic inactivation than similar modification of Lys-55 in K59Q. The results in sum show that, though Lys-55 and Lys-59 may be at or near the active site, neither is indispensable for the catalytic activity of HMBS.
摘要
将携带hemC基因的新构建体转化到大肠杆菌中，导致羟甲基胆色素原合酶（HMBS）的表达量约为原来的1000倍。该构建体用于生成HMBS，其中（a）第55位赖氨酸、（b）第59位赖氨酸以及（c）第55位和第59位赖氨酸均被谷氨酰胺取代（分别为K55Q、K59Q和K55Q - K59Q）。所有这三种修饰后的酶在色谱上均可与野生型酶分离。动力学研究表明，K55Q取代对酶活性影响不大，而K59Q取代导致表观催化常数（Kapp. cat.）与表观米氏常数（Kapp. m）的比值降低25倍。分别用磷酸吡哆醛和硼氢化钠处理K55Q、K59Q和K55Q - K59Q，结果发现与剩余赖氨酸残基发生了不完全且非特异性的反应。K55Q突变体中第59位赖氨酸的吡哆醛修饰比K59Q中第55位赖氨酸的类似修饰导致更大程度的酶失活。总体结果表明，尽管第55位和第59位赖氨酸可能位于活性位点或其附近，但两者对于HMBS的催化活性都不是不可或缺的。
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