Adenylosuccinate Lyase: A Study of the Novel Intersubunit Active Sites

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Adenylosuccinate lyase catalyzes a critical step in purine biosynthesis by converting adenylosuccinate to AMP and fumarate. High-resolution structures of two bacterial enzymes in the absence of substrate have been reported. Three histidines, contributed by two different subunits, have been identified in the active site. However, the structure of adenylosuccinate lyase suggests that in an enzyme tetramer, three subunits actually contribute to each of the four active sites. This project's goal is to understand the major chemical and structural contributions to catalysis by normal adenylosuccinate lyase and the molecular basis for decreased activity in patients with ASL deficiency. The specific aims address three questions: 1. Which additional amino acids of adenylosuccinate lyase contribute to catalysis and/or substrate binding? This issue is approached by site-directed mutagenesis of amino acids postulated as at the active site of B. subtilis adenylosuccinate lyase. Stable analogs of adenylosuccinate, inert to enzymatic cleavage, will be synthesized and used to prepare crystalline enzyme-inhibitor complexes; they will also be used to assess substrate binding by enzymes. 2. How many subunits contribute to each active site of adenylosuccinate lyase? Complementation experiments will be conducted in which pairs of different, inactive mutants are mixed and tested for restoration of activity in hybrid tetramers. 3. What is the molecular basis for the structure-function relations in the adenylosuccinate lyase? B. subtilis adenylosuccinate lyase will be engineered with amino acid substitutions equivalent to those in some human variants with adenylosuccinate lyase deficiency. These mutant enzymes will be models to elucidate further the structural/chemical basis of the enzyme function. (This project is supported jointly by the Molecular Biochemistry and Metabolic Biochemistry Programs.)