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

本研究的目的是描述假单胞菌脂肪酶和酯解抗体两种催化相关蛋白的反应机制和作用方式，以及它们在制备分子转化中的应用。制备了针对酯水解的膦酸过渡态类似物的单克隆抗体。其中一种抗体被发现催化水解具有高立体选择性的外消旋酯。然而，由于强烈的产物抑制作用，该反应几乎是化学计量的。对该抗体进行的一系列化学修饰表明，抗体结合位点上的一个Arg残基对该抗体的活性至关重要。事实上，通过克隆编码可变结构域的基因，在抗体结合位点（CDR3）上发现了一个精氨酸。同样的精氨酸还被发现在与带负电荷的产物的电荷相互作用中起主导作用，因为抗体与碳酸酯的产物抑制作用要小得多，碳酸酯在水解时脱羧，产生中性醇作为最终产物。该抗体表现出至少100次周转，而与该底物的活性没有任何损失。从假单胞菌中克隆出一种编码脂肪酶的基因，并在大肠杆菌中大量产生。然而，脂肪酶以包涵体的非活性形式产生。我们发现了另一个位于脂肪酶基因后的基因（lip B），并在大肠杆菌中克隆并过量生产了该基因，发现其产生的蛋白具有伴侣蛋白样活性，有助于在体外对变性脂肪酶进行重折叠，从而恢复脂肪酶的活性。lipb的作用是亲本脂肪酶所特有的，这表明它是假单胞菌脂肪酶的一个私人伴侣蛋白。

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.