通过重新设计乳酸脱氢酶框架获得一种特异性高活性苹果酸脱氢酶。

A specific, highly active malate dehydrogenase by redesign of a lactate dehydrogenase framework.

作者信息

Wilks H M, Hart K W, Feeney R, Dunn C R, Muirhead H, Chia W N, Barstow D A, Atkinson T, Clarke A R, Holbrook J J

机构信息

Department of Biochemistry, University of Bristol, United Kingdom.

出版信息

Science. 1988 Dec 16;242(4885):1541-4. doi: 10.1126/science.3201242.

DOI:10.1126/science.3201242

PMID:3201242

Abstract

Three variations to the structure of the nicotinamide adenine dinucleotide (NAD)-dependent L-lactate dehydrogenase from Bacillus stearothermophilus were made to try to change the substrate specificity from lactate to malate: Asp197----Asn, Thr246----Gly, and Gln102----Arg). Each modification shifts the specificity from lactate to malate, although only the last (Gln102----Arg) provides an effective and highly specific catalyst for the new substrate. This synthetic enzyme has a ratio of catalytic rate (kcat) to Michaelis constant (Km) for oxaloacetate of 4.2 x 10(6)M-1 s-1, equal to that of native lactate dehydrogenase for its natural substrate, pyruvate, and a maximum velocity (250 s-1), which is double that reported for a natural malate dehydrogenase from B. stearothermophilus.

摘要

为了尝试将嗜热脂肪芽孢杆菌烟酰胺腺嘌呤二核苷酸（NAD）依赖性L-乳酸脱氢酶的底物特异性从乳酸改变为苹果酸，对其结构进行了三种改造：天冬氨酸197突变为天冬酰胺、苏氨酸246突变为甘氨酸、谷氨酰胺102突变为精氨酸。尽管只有最后一种改造（谷氨酰胺102突变为精氨酸）为新底物提供了一种高效且高度特异性的催化剂，但每种改造都使特异性从乳酸转变为苹果酸。这种合成酶对草酰乙酸的催化速率（kcat）与米氏常数（Km）之比为4.2×10⁶M⁻¹ s⁻¹，与天然乳酸脱氢酶对其天然底物丙酮酸的该比值相同，并且其最大反应速度为250 s⁻¹，是报道的嗜热脂肪芽孢杆菌天然苹果酸脱氢酶最大反应速度的两倍。