SITE DIRECTED MUTAGENESIS OF HISTIDINE DECARBOXYLASE

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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.