Adenylosuccinate Lyase: Novel Intersubunit Active Sites

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DESCRIPTION (provided by applicant): Adenylosuccinate lyase catalyzes 2 distinct but chemically related steps in purine biosynthesis: the conversion of adenylosuccinate to AMP + fumarate, and the cleavage of 5-aminoimidazole-4-(N-succinylocarboxamide)ribonucleotide (SAICAR) to 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) + fumarate. The importance of the metabolic role played by adenylosuccinate lyase (ASL) is indicated by the severity of the symptoms of ASL deficiency in humans; the disease is associated with mental retardation, psychomotor delay, epilepsy and autism. ASL is isolated as a tetramer of identical subunits. Crystals of enzyme in the absence of substrate or inhibitor have been prepared from 3 bacterial species and high-resolution structures have been reported for two of them. In Dr. Colman's laboratory, affinity labeling and site-directed mutagenesis of the Bacillus subtilis ASL led to the identification of 3 His (1168, H89 and H141) as critical for catalysis, 2 of which likely function as general acid/general base during the reaction and are contributed by 2 different subunits. However, examination of the structure of adenylosuccinate lyase leads to the hypothesis that in an enzyme tetramer, 3 subunits provide amino acids to each of the 4 active sites. The overall goal of this project is to understand the major chemical and structural contributions to catalysis by normal ASL and the molecular basis for decreased activity in patients with ASL deficiency. Following questions are being now asked: which additional amino acids of ASL participate in catalysis and/or substrate binding, and which subunits provide these residues? These issues will be approached by site-directed mutagenesis of amino acids of B. subtilis ASL postulated to be at the active site. Targets of mutagenesis will be selected on the basis of conservation among the sequences of ASL from 28 species and proximity in the crystal structures to the active site. Mutant enzymes will be expressed, purified to homogeneity and extensively characterized by kinetics, binding and biophysical measurements. Complementation experiments will be conducted in which pairs of different, inactive mutants are mixed and tested for restoration of activity in hybrid tetramers. Bifunctional affinity labels will be evaluated to ascertain whether crosslinking of subunits occurs. To elucidate the molecular basis of ASL deficiency, mutants of B. subtilis ASL will be constructed with amino acid substitutions equivalent to those in human patients with ASL deficiency. The mutant enzymes will be purified and extensively characterized. The intention is to use these mutant enzymes as models to evaluate the structural/chemical basis of a human genetic defect.