Department of Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo, CA 93407, USA.
Biochemistry. 2010 Aug 3;49(30):6485-93. doi: 10.1021/bi100648w.
Pyridoxal phosphate (PLP)-dependent enzymes catalyze many different types of reactions at the alpha-, beta-, and gamma-carbons of amine and amino acid substrates. Dialkylglycine decarboxylase (DGD) is an unusual PLP-dependent enzyme that catalyzes two reaction types, decarboxylation and transamination, in the same active site. A structurally based, functional model has been proposed for the DGD active site, which maintains that R406 is important in determining substrate specificity through interactions with the substrate carboxylate while W138 provides specificity for short-chain alkyl groups. The mechanistic roles of R406 and W138 were investigated using site-directed mutagenesis, alternate substrates, and analysis of steady-state and half-reaction kinetics. Experiments with the R406M and R406K mutants confirm the importance of R406 in substrate binding. Surprisingly, this work also shows that the positive charge of R406 facilitates catalysis of decarboxylation. The W138F mutant demonstrates that W138 indeed acts to limit the size of the subsite C binding pocket, determining specificity for 2,2-dialkylglycines with small side chains as predicted by the model. Finally, work with the double mutant W138F/M141R shows that these mutations expand substrate specificity to include l-glutamate and lead to an increase in specificity for l-glutamate over 2-aminoisobutyrate of approximately 8 orders of magnitude compared to that of wild-type DGD.
磷酸吡哆醛（PLP）依赖性酶在胺和氨基酸底物的α、β和γ位催化许多不同类型的反应。二烃基甘氨酸脱羧酶（DGD）是一种不寻常的 PLP 依赖性酶，在同一个活性位点中催化脱羧和转氨两种反应类型。已经提出了一种基于结构的、功能性的 DGD 活性位点模型，该模型认为 R406 通过与底物羧酸的相互作用在决定底物特异性方面很重要，而 W138 为短链烷基基团提供特异性。通过定点突变、替代底物以及稳态和半反应动力学分析，研究了 R406 和 W138 的作用机制。与 R406M 和 R406K 突变体的实验证实了 R406 在底物结合中的重要性。令人惊讶的是，这项工作还表明，R406 的正电荷有助于促进脱羧反应的催化。W138F 突变体表明，W138 确实起到了限制亚位点 C 结合口袋大小的作用，这与模型预测的小侧链 2,2-二烃基甘氨酸的特异性一致。最后，与双突变体 W138F/M141R 的工作表明，这些突变使底物特异性扩展到包括 l-谷氨酸，并导致与野生型 DGD 相比，对 l-谷氨酸的特异性增加约 8 个数量级，超过 2-氨基异丁酸。
