Bacillus subtilis adenylosuccinate lyase: I. Identification of enzyme active site residues. II. Model system for elucidating the biochemistry of human acenylosuccinate lyase deficiency

Bacillus subtilis adenylosuccinate lyase is a tetrameric enzyme participating twice in the purine biosynthetic pathway. The reactions catalyzed proceed by a β-elimination mechanism, and His68 and His141 serve as the general acid and base, respectively [Lee et. a1. (1999) Biochemistry 38: 22–32] With the goal of identifying additional active site residues, sequence alignments and homology modeling, based on the crystal structure of Thermotoga maritima adenylosuccinate lyase, were used to select target amino acids. The roles of these residues were subsequently defined through site directed mutagenesis, kinetic characterization, and biophysical studies. One of three conserved histidines, His89 interacts with the AMP portion of substrate. Lys268, part of a conserved signature sequence region, contributes to substrate binding through its close interaction with the phosphoryl group. Glu275, also in the signature sequence region, properly orients His141 for effective catalysis.; Enzyme subunit complementation experiments were also conducted, in which pairs of mutant enzymes were mixed to form hybrid enzymes with restoration of activity, revealing the subunits which contribute residues to the enzyme active site. His141 is contributed from a subunit distinct from that contributing His68 and His89. Lys 268 and Glu275 come from a third, separate subunit, providing the first functional evidence that three subunits contribute residues to each enzyme active site.; This enzyme is also associated with a human disease, adenylosuccinate lyase deficiency. The deficiency arises from several single point mutations in the gene encoding the enzyme, resulting in mild to severe mental retardation, muscle wasting, and autistic features. With the goal of elucidating the underlying biochemistry associated with this human disease, four mutant enzymes, corresponding to mild and severe forms of the deficiency, were constructed and characterized (M26L, R141W, R303C, and S395R, human enzyme numbering). Bacillus subtilis adenylosuccinate lyase was used a model system in which to reconstruct these mutations. Biophysical characterization revealed these mutant enzymes are associated with marked thermal instability and structural aberrations. These particular residues do not play catalytic roles; rather they are in regions key for enzyme stability and structural integrity.