Chang K H, Xiang H, Dunaway-Mariano D
Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, USA.
Biochemistry. 1997 Dec 16;36(50):15650-9. doi: 10.1021/bi971262p.
4-Chlorobenzoate:coenzyme A (4-CBA:CoA) ligase catalyzes 4-chlorobenzoyl-coenzyme A formation in a two-step reaction consisting of the adenylation of 4-chlorobenzoate with adenosine 5'-triphosphate followed by acyl transfer from the 4-chlorobenzoyl adenosine 5'-monophosphate diester intermediate to coenzyme A. In this study, two core motifs present in the Pseudomonas sp. strain CBS3 4-CBA:CoA ligase (motif I, 161T-S-G-T-T-G-L-P-K-G170, and motif II, 302Y-G-T-T-E306) and conserved among the sequences representing the acyl-adenylate/thioester-forming enzyme family (to which the ligase belongs) were tested for their possible role in substrate binding and/or catalysis. The site-directed mutants G163I, G166I, P168A, K169M, and E306Q were prepared and then subjected to steady-state and transient kinetic studies. The results, which indicate reduced catalysis of the adenylation of 4-chlorobenzoate in the mutant enzymes, are interpreted within the context of the three-dimensional structure of the acyl-adenylate/thioester-forming enzyme family member, firefly luciferase.
4-氯苯甲酸：辅酶A（4-CBA:CoA）连接酶通过两步反应催化生成4-氯苯甲酰辅酶A，该反应包括4-氯苯甲酸与三磷酸腺苷的腺苷化反应，随后是4-氯苯甲酰腺苷5'-单磷酸二酯中间体的酰基转移至辅酶A。在本研究中，对假单胞菌属菌株CBS3的4-CBA:CoA连接酶中存在的两个核心基序（基序I，161T-S-G-T-T-G-L-P-K-G170，和基序II，302Y-G-T-T-E306）进行了测试，这些基序在代表酰基腺苷酸/硫酯形成酶家族（连接酶所属家族）的序列中保守，以研究它们在底物结合和/或催化中的可能作用。制备了定点突变体G163I、G166I、P168A、K169M和E306Q，然后进行稳态和瞬态动力学研究。结果表明突变酶中4-氯苯甲酸腺苷化的催化作用降低，这些结果在酰基腺苷酸/硫酯形成酶家族成员萤火虫荧光素酶的三维结构背景下进行了解释。
