PROTEIN ENGINEERING OF HISTIDINE DECARBOXYLASE

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The overall goal of this proposed research is to further our understanding of the mechanisms of enzyme action, and to help define rules for protein design. The object of study is histidine decarboxylase (HDC) from Lactobacillus 30a. This unusual protein undergoes an autoactivation step forming its pyruvoyl cofactor. It also exhibits cooperative kinetics and appears to possess a vectorial substrate flow system for substrate and product. Five classes of site directed mutations will be made in HDC, and will be based on our understanding of the structure and kinetics of the enzyme. 1) Active site residues like Ile 59, Tyr 62, and Asp 63 are known to interact with substrate and will be altered to quantify the importance of those interactions. These and other residues may also participate in the cooperative kinetics seen for HDC, a phenomenon investigated initially by mutagenesis of another "cross boundary" residue, Glu 66. In addition more dramatic alterations, and pairs of changes, will be made to give a clearer view of key residues and their interactions. 2) It is suspected that cationic histidine is guided into a central active site well in the catalytic trimer by an electrostatic field effect. This will be tested by perturbing several carboxylates ringing the well which are suspected to create the field. Initially amides will replace the carboxylates, but positive residues may also be introduced. 3) The x-ray structure suggests that HDC may in fact possess a substrate flow system, in which substrate enters from the central well mentioned above, react at the pyruvoyl site, and the product exists through a water filled tunnel which runs from the back of the catalytic site, through the trimer wall to the outside. Mutations will be made to test this hypothesis by blocking the tunnel in various ways. 4) Efforts will be made to alter the topography of the active site cleft. Mutations will be made which will aim to alter substrate specificity of HDC, perhaps allowing it to act on ornithine, lysine or asparagine. Also, efforts will be made to adjust the catalytic site to accommodate a novel alpha-ketobutyroyl cofactor by converting Tyr 262 to a smaller Leu residue. 5) Mutations will be made to alter the HDC conformation. One kind will disrupt the hexameric structure and produce trimers. Another will aim to relieve the folding strain which drive autoactivation.