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Studies on the mechanisms of substrate recognition and enzyme action in aspartate aminotransferase

天冬氨酸转氨酶底物识别和酶作用机制的研究

基本信息

批准号：
04454160
负责人：
KAGAMIYAMA Hiroyuki
金额：
$ 4.67万
依托单位：
Osaka Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1994
项目状态：
已结题

项目摘要

We have examined the functional role of some active site residues of E.coli aspartate, aromatic, and branched amino acid aminotransferases. Our findings are as follows :1. artate aminotransferases (AspAT).1) The negative charge on Asp222 stabilyzes the positive charge on N (1) of the coenzyme, pyridoxal phosphate, and aids the abstraction of an alpha-proton from the substrate during the catalysis. Asp222 is also important for supporting the coenzyme ring in position.2) Asn194 functions in substrate binding through hydrogen bonding to the substrate carboxylate and/or holding the side chain of Arg386, which interacts with the substrate alpha-carboxylate, in a position suitable for substrate binding. Further, we propose that the idea that Asn194 lowers the pKa of the imine nitrogen of the internal aldimine bond to facilitate a transaldimination step.3) The X-ray crystallography revealed the conformational change upon binding of the substrate (inhibitor) to close the active site. At the sa … More me time, neutralization of the positive charge on these two arginine residues increases the pKa of the internal aldimine through direct Coulombic interaction and Arg386-Asn194-coenzyme hydrogen bonding network, facilitating transaldimination step.2. Aromatic amino acid aminotransferase (arAT).ArAT from E.coli was overexpressed in E.coli cells, purified, and characterized. ArAT and AspAT showed overlapping substrate specificity. Both of the enzymes were active toward dicarboxylic substrates. However, ArAT showed 10^3-fold higher activity toward aromatic substrates than AspAT,and this was in part ascribed to the active site hydrophobicity. Asn194 and Arg386 of ArAT had similar effect on the pKa of aldimine as observed for AspAT.3. Branched-amino acid aminotransferase (BrAT).Preliminary X-ray characterization of BrAT was achieved. BrAT catalyzes pro-R C-4' hydrogen transfer through the coenzyme-substrate Schiff base intermediate as observed in D-amino acid aminotransferase, in contrast to other various aminotransferases catalysing the pro-S hydrogen transfer. Less

我们已经研究了大肠杆菌天冬氨酸，芳香族和支链氨基酸氨基转移酶的一些活性位点残基的功能作用。研究结果如下：1. 1）Asp 222上的负电荷稳定辅酶磷酸吡哆醛的N（1）上的正电荷，并有助于在催化过程中从底物中提取α-质子。Asp 222对于将辅酶环支撑在适当的位置也很重要。2）Asn 194通过与底物羧酸盐的氢键结合和/或将与底物α-羧酸盐相互作用的Arg 386的侧链保持在适合于底物结合的位置而在底物结合中起作用。此外，我们认为Asn 194降低了内部醛亚胺键的亚胺氮的pKa，以促进transaldimination步骤。3）X射线晶体学揭示了底物（抑制剂）结合后的构象变化，以关闭活性位点。在SA ...更多信息同时，这两个精氨酸残基上的正电荷的中和通过直接库仑相互作用和Arg 386-Asn 194-辅酶氢键网络增加了内部醛亚胺的pKa，促进了transaldimination步骤。芳香族氨基酸氨基转移酶（arAT）：来自大肠杆菌的ArAT在大肠杆菌细胞中过表达，纯化并表征。ArAT和AspAT显示重叠的底物特异性。这两种酶对二羧酸底物都有活性。然而，ArAT对芳香底物的活性比AspAT高10^3倍，这部分归因于活性位点的疏水性。ArAT的Asn 194和Arg 386对醛亚胺的pKa具有与AspAT相似的影响。支链氨基酸氨基转移酶（BrAT）：初步X射线表征的BrAT。与催化pro-S氢转移的其他各种氨基转移酶相比，BrAT催化pro-R C-4'氢转移通过辅酶-底物席夫碱中间体，如在D-氨基酸氨基转移酶中观察到的。少