Tadrowski Sonya, Pedroso Marcelo M, Sieber Volker, Larrabee James A, Guddat Luke W, Schenk Gerhard
School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, QLD 4072, Australia.
Straubing Center of Science, Technische Universität München, Straubing, Germany.
Chemistry. 2016 May 23;22(22):7427-36. doi: 10.1002/chem.201600620. Epub 2016 May 2.
Ketol-acid reductoisomerase (KARI) is a Mg(2+) -dependent enzyme in the branched-chain amino acid biosynthesis pathway. It catalyses a complex two-part reaction: an alkyl migration followed by a NADPH-dependent reduction. Both reactions occur within the one active site, but in particular, the mechanism of the isomerisation step is poorly understood. Here, using a combination of kinetic, thermodynamic and spectroscopic techniques, the reaction mechanisms of both Escherichia coli and rice KARI have been investigated. We propose a conserved mechanism of catalysis, whereby a hydroxide, bridging the two Mg(2+) ions in the active site, initiates the reaction by abstracting a proton from the C2 alcohol group of the substrate. While the μ-hydroxide-bridged dimetallic centre is pre-assembled in the bacterial enzyme, in plant KARI substrate binding leads to a reduction of the metal-metal distance with the concomitant formation of a hydroxide bridge. Only Mg(2+) is capable of promoting the isomerisation reaction, likely to be due to non-competent substrate binding in the presence of other metal ions.
酮醇酸还原异构酶（KARI）是支链氨基酸生物合成途径中一种依赖Mg(2+)的酶。它催化一个复杂的两步反应：首先是烷基迁移，随后是依赖NADPH的还原反应。这两个反应都在同一个活性位点内发生，但具体而言，异构化步骤的机制仍知之甚少。在此，通过结合动力学、热力学和光谱技术，对大肠杆菌和水稻的KARI的反应机制进行了研究。我们提出了一种保守的催化机制，即一个桥接活性位点中两个Mg(2+)离子的氢氧根，通过从底物的C2醇基团夺取一个质子来启动反应。虽然在细菌酶中μ-氢氧根桥连的双金属中心是预先组装好的，但在植物KARI中，底物结合会导致金属-金属距离缩短，并伴随氢氧根桥的形成。只有Mg(2+)能够促进异构化反应，这可能是由于在存在其他金属离子的情况下底物结合不充分所致。
