RACEMIZATION OF ALANINE BY THE ALANINE RACEMASES FROM SALMONELLA-TYPHIMURIUM AND BACILLUS-STEAROTHERMOPHILUS - ENERGETIC REACTION PROFILES
RACEMIZATION OF ALANINE BY THE ALANINE RACEMASES FROM SALMONELLA-TYPHIMURIUM AND BACILLUS-STEAROTHERMOPHILUS - ENERGETIC REACTION PROFILES
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DOI：
10.1021/bi00409a022
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发表时间：
1988-05-03
期刊：
影响因子：
2.9
通讯作者：
WALSH, CT
中科院分区：
文献类型：
--
作者：
FARACI, WS;WALSH, CT
丙氨酸外消旋酶是细菌吡哆醛5' -磷酸（PLP）依赖性酶，提供d -丙氨酸作为细胞壁肽聚糖层生物合成的重要组成部分。研究了来自革兰氏阴性嗜温性鼠伤寒沙门菌和革兰氏阳性嗜热性脂嗜热芽孢杆菌的两个同工酶丙氨酸外消旋酶的外消旋机制，分别由dadB基因和alr基因编码。在L .fwdarw中测量了底物氘同位素效应和溶剂氘同位素效应。D和D。所有三种酶的L方向，以评估提取的程度。-质子或底物PLP碳的质子化在催化中是有限的。此外，实验测量的内部回报。每种酶在3H2O条件下从底物到产物的-3H和溶剂交换/底物转化实验都被用来检测底物PLP碳中间体的分配，并获得丙氨酸消旋酶催化中具有动力学意义的能垒的相对高度。
Alanine racemases are bacterial pyridoxal 5''-phosphate (PLP) dependent enzymes providing D-alanine as an essential building block for biosynthesis of the peptidoglycan layer of the cell wall. Two isozymic alanine racemases, encoded by the dadB gene and the alr gene, from the Gram-negative mesophilic Salmonella typhimurium and one from the Gram-positive thermophlic Bacillus stearothermophilus have been examined for the racemization mechanism. Substrate deuterium isotope effects and solvent deuterium isotope effects have been measured in both L .fwdarw. D and D .fwdarw. L directions for all three enzymes to assess the degree to which abstraction of the .alpha.-proton or protonation of substrate PLP carbanion is limiting in catalysis. Additionally, experiments measuring internal return of .alpha.-3H from substrate to product and solvent exchange/substrate conversion experiments in 3H2O have been used with each enzyme to examine the partitioning of substrate PLP carbanion intermediates and to obtain the relative heights of kinetically significant energy barriers in alanine racemase catalysis.