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Reaction Mechanisms and Mode of Action of a Catalytic Antibody and Pseudomonas Lipase

催化抗体与假单胞菌脂肪酶的反应机制和作用方式

基本信息

批准号：
05453167
负责人：
ODA Junichi
金额：
$ 4.29万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

The purpose of this study is to delineate the reaction mechanisms and mode of action of two catalytically-related proteins-a Pseudomonas lipase and an esterolytic antibody, and their applications to preparative molecular transformations.Monoclonal antibodies were generated against a phosphonate transitionstate analogue for ester hydrolysis.One of the antibodies was found to catalyze the hydrolysis of a racemic ester with high stereoselsecitivy. The reaction, however, was almost stoichiometric due to strong product inhibition. A series of chemical modification of this antibody revealed that one Arg residue in the antibody combining site was essential to the activity.In fact, one Arg was found in the antibody combining site (CDR3) by cloning the gene encoding the variable domain. The same Arg was also found to play a dominant role in product inhibition by charge interaction with a negatively charged product, because the antibody experienced much less product inhibition with a carbonate ester, which undergoes decarboxylation upon hydrolysis to yield a neutral alcohol as the final product. The antibody exhibited at least 100 turnovers without any loss of activity with this substrate.A gene encoding a lipase was cloned from a Pseudomonas sp.and was overproduced in E.coli. The lipase, however, was produced as a non-active form of inclusion bodies. We found another gene (lip B) located right after the lipase gene, and cloned and overproduced the gene in E.coli.The hitherto unknown protein produced was found to have a chaperon-like activity, assiting the refolding of a denatured lipase in vitro to restore the lipase activity. The action of Lip B was unique to the parent lipase, suggesting a private chaperonin to the Pseudomonas lipase.

本研究的目的是描述两种催化相关蛋白--假单胞菌脂肪酶和酯水解抗体的反应机理和作用模式，以及它们在制备性分子转化中的应用.制备了抗磷酸酯过渡态类似物酯水解的单克隆抗体，发现其中一种抗体催化具有高立体选择性的外消旋酯的水解.然而，由于强烈的产物抑制，反应几乎是化学计量的。对该抗体进行了一系列化学修饰，发现抗体结合位点上有一个Arg残基对该抗体的活性至关重要，实际上，通过克隆编码可变区的基因，在抗体结合位点（CDR 3）上发现了一个Arg残基。还发现相同的Arg通过与带负电荷的产物的电荷相互作用在产物抑制中起主导作用，因为抗体经历碳酸酯的少得多的产物抑制，碳酸酯在水解时经历脱羧以产生中性醇作为最终产物。该抗体在此底物下至少有100次的转化而没有任何活性损失。从假单胞菌中克隆了一个编码脂肪酶的基因，并在大肠杆菌中过量生产。然而，脂肪酶是作为非活性形式的包涵体产生的。我们发现另一个基因（lip B）位于脂肪酶基因之后，并克隆了该基因，在大肠杆菌中进行了过量生产，发现所产生的蛋白具有伴侣样活性，有助于变性脂肪酶在体外的重折叠以恢复脂肪酶活性。Lip B的作用是亲本脂肪酶所特有的，表明它是假单胞菌脂肪酶的私人伴侣。