SITE DIRECTED MUTAGENESIS OF HISTIDINE DECARBOXYLASE

来自乳酸杆菌的组氨酸脱羧酶（HDC）催化组氨酸脱羧， 反应组氨酸-大于组胺+ CO2。 这 一种特殊的酶以非活性形式合成， 通过Ser 81和Ser 82之间的肽键的切割而产生。 作为该过程的一部分，Ser 82被转化为Pyr， 作为脱羧的酶辅因子。 化学研究表明，几种常见的酸和碱 必须参与自动激活和催化过程; X射线研究提出了几个候选人，基于他们的 接近活性部位。 我们已经克隆并测序了HDC的基因， 激活突变体 该蛋白已在E.杆菌 从质粒，我们建议进行我们的计划， 寡核苷酸定向的位点特异性诱变HDC。 我们 目的是分析各种残留物对这两种物质的贡献， 酶的自激活机制和催化机理。 在要进行的突变中，有Ser 82转化为 Cys和Thr两者。丝氨酸81是丙酮酸中的一个保守残基 需要脱羧酶来稳定自激活 中间体，将转化为Ala。Lys 155，可以 结合底物羧基，将转化为Gln。 在 此外，三个酸性基团，Glu 197，Glu 66和Asp 63， 在活性位点附近的化合物将被改变，最初为酰胺，而 两个Tyr基团，62和262，其可在自活化中起作用， 首先转化为Phe。

Histidine decarboxylase (HDC) from Lactobacillus catalyzes the reaction histidine --- greater than histamine + CO2. This particular enzyme is synthesized in an inactive form and activates itself by cleavage of the peptide bond between Ser 81 and Ser 82. As part of this process, Ser 82 is converted to a pyr which serves as the enzymatic cofactor for the decarboxylation. Chemical studies have shown that several general acids and bases must be involved in the auto-activation and catalytic processes; X-ray studies have suggested several candidates, based on their proximity to the active site. We have cloned and sequenced the genes for HDC and one activation mutant. The proteins have been expressed in E. coli from plasmids and we propose to carry our a program of oligonucleotide directed site specific mutagenesis of HDC. We aim to analyze the contribution of various residues to both the auto-activation scheme and catalytic mechanism of the enzyme. Among the mutations to be made are the conversion of Ser 82 to both Cys and Thr. Ser 81, a conserved residue in pyruvate requiring decarboxylases which may stabilize the auto-activation intermediate, will be converted to an Ala. Lys 155, which may bind the substrate carboxyl, will be converted to an Gln. In addition, three acidic groups, Glu 197, Glu 66 and Asp 63, which are near the active site will be altered, initially to amides, while two Tyr groups, 62 and 262, which may act in auto-activation, will initially be converted to Phe.