Enzyme Structure-Function Relationships

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9418479 Remington X-ray crystallographic and mutagenesis studies will be done on two enzymes, citrate synthase from pig and Azotobacter and serine carboxypeptidase from wheat and yeast. Atomic models for the pig, wheat and yeast enzymes are on hand and have been refined at high resolution . These will be used as the basis for inhibitor binding studies that will probe several aspects for the catalytic mechanism. Inhibitors have been designed that mimic proposed transition states of various steps along the catalytic pathway, so as to provide "snapshots: of enzyme conformations and interactions with substrate along the catalytic pathway. The yeast serine carboxypeptidase has been mutated, resulting in greatly changed substrate specificity, and binding studies with peptide aldehydes should reveal the structural consequences of the mutational change and the molecular basis for substrate specificity. The structure of the Azotobacter enzyme will be determined in order to study how bacterial citrate synthases are allosterically regulated by NADH, and how they differ in structure from the non-allosterically regulated enzyme from pig. The bacterial enzyme will also be used as a vehicle for mutagenesis studies which will be designed to probe the functions of specific active-site amino acid side chains. The impetus for this is that most of many interesting mutants of the pig enzyme that exist cannot be crystallized, so the structural consequences of the mutations are unknown. These projects are expected to shed light on the enzymatic mechanisms for two different families of enzymes. Each enzyme poses significant problems concerning the mechanism of catalysis that remain to be understood. Citrate synthase is the only enzyme of known structure that can form a carbon-carbon bond. The energetics of the initial step in the reaction, the deprotonation of a carbon acid, are not understood. Serine carboxypeptidases are serine proteinases that function optimally at pH 4.0-5.0, unlike the other serine proteinases, and the structural basis for this is also not understood. %% X-ray crystallographic and mutagenesis studies will be done on two enzymes, citrate synthetase from pig and Azotobacter, and serine carboxypeptidase from wheat and yeast. These projects are expected to shed light on the enzymatic mechanisms for the two different families of enzymes. Each enzyme possesses significant problems concerning the mechanism of catalysis that remain to be understood. The structure of the Azotobacter enzyme will be determined in order to study how these bacterial enzymes are regulated and how they differ from the pig enzyme. The bacterial enzyme will also be mutated to probe the function of specific active-site amino acid side chains. The yeast serine carboxypeptidase has been mutated, resulting in greatly changed substrate specificity. Binding studies with substrate analogs should reveal the structural consequences of the mutational change and the molecular basis of substrate specificity. ***