Faraci W S, Walsh C T
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
Biochemistry. 1988 May 3;27(9):3267-76. doi: 10.1021/bi00409a022.
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 thermophilic Bacillus stearothermophilus have been examined for the racemization mechanism. Substrate deuterium isotope effects and solvent deuterium isotope effects have been measured in both L----D and D----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.
丙氨酸消旋酶是细菌中依赖于磷酸吡哆醛（PLP）的酶，它能提供D-丙氨酸，作为细胞壁肽聚糖层生物合成的必需组成部分。已对来自革兰氏阴性嗜温鼠伤寒沙门氏菌的由dadB基因和alr基因编码的两种同工型丙氨酸消旋酶，以及来自革兰氏阳性嗜热嗜热脂肪芽孢杆菌的一种丙氨酸消旋酶的消旋机制进行了研究。已在L→D和D→L两个方向上对所有这三种酶测量了底物氘同位素效应和溶剂氘同位素效应，以评估α-质子的夺取或底物PLP碳负离子的质子化在催化过程中的限制程度。此外，已对每种酶进行了测量α-3H从底物到产物的内部回返的实验，以及在3H2O中进行的溶剂交换/底物转化实验，以研究底物PLP碳负离子中间体的分配情况，并获得丙氨酸消旋酶催化过程中动力学上显著的能量屏障的相对高度。
