SITE DIRECTED MUTAGENESIS OF HISTIDINE DECARBOXYLASE

乳酸杆菌的组氨酸脱羧酶(HDC)催化 组氨酸反应-大于组胺+二氧化碳。这 特定的酶以不活跃的形式合成，并被激活 通过裂解丝氨酸81和丝氨酸82之间的多肽键。 作为该过程的一部分，Ser82被转换为PYR 作为脱羧酶的辅因子。 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光研究已经提出了几个候选人，基于他们的 离活动地点很近。 我们已经对HDC和One的基因进行了克隆和测序 激活突变体。这些蛋白已在大肠杆菌中表达。 我们计划实施我们的一个项目 寡核苷酸引导HDC的定点突变。我们 目的分析不同残留物对生物多样性的贡献。 该酶的自动激活方案和催化机理。 将进行的突变包括将Ser82转换为 赛斯和苏尔都是。丙酮酸中的保守残基Ser81 需要可以稳定自激活的脱羧酶 中级，将被转换为ALA。Lys 155，可能 结合底物羧基，会转化为谷氨酰胺。在……里面 此外，谷氨酸197、谷氨酸66和天冬氨酸63三个酸性基团 在活性中心附近将被改变，最初为酰胺，而 可以自动激活的两个TYR组62和262将 最初将转换为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.