Studies on the bacterial glycogen biosynthetic enzymes will be continued with respect to elucidating their structure and to relate structure with function and specificity of binding at various ligand binding sites (e.g., inhibitor, activator and catalytic sites). These studies will be centered on the enzyme, ADPglucose pyrophosphorylase. A study of kinetically altered ADPglucose pyrophosphorylases from E. coli glycogen mutants is initiated to determine the relationship of allosteric function with the primary structure of the ADPglucose pyrophosphorylase. The cloning of the structural gene for the ADPglucose pyrophosphorylase of the E. coli allosteric mutant, 618, and the nucleotide sequence studies of it will enable us to determine the amino acid substitution and give us some insight in the study of structure and function of the allosteric site(s). Other E. coli ADPglucose pyrophosphorylase allosteric mutants that have been previously described will also be cloned to provide more information on the structure and function of the allosteric site(s). Cloning of the structural genes of the ADPglucose pyrophosphorylase from other bacteria (e.g., R. rubrum, R. capsulata, R. sphaeroides) are planned and attempts to obtain good expression of the gene are also planned. Nucleotide sequence studies and covalent modification of the various proteins with activator analogues may provide us with useful information of the sequence of the allosteric sites of these ADPglucose pyrophosphorylases. These comparative studies at the protein sequence level will enable us to relate structure with function and activator specificity at these various ligand binding sites. The characterization of E. coli B glycogen synthase and branching enzyme with respect to reaction mechanism and the various amino acids involved in catalysis will be continued. Studies on the cloning of the glycogen biosynthetic structural genes of E. coli K12 will be continued to enable us to determine DNA sequence analysis of the glg (glycogen synthase) structural gene. From this analysis the amino acid sequence will be deduced. The genetic regulation of the biosynthesis of the glycogen biosynthetic enzymes will also be studied and it is hoped that the isolation of the glg genes from both E. coli and Salmonella typhimurium will provide us with some insight in the mode of genetic regulation of expression of the glg genes.
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