MOTIFS AND STRUCTURAL FOLD OF THE COFACTOR BINDING-SITE OF HUMAN GLUTAMATE-DECARBOXYLASE

The pyridoxal-P binding sites of the two isoforms of human glutamate d ecarboxylase (GAD65 and GAD67) were modeled by using PROBE (a recently developed algorithm for multiple sequence alignment and database sear ching) to align the primary sequence of GAD with pyridoxal-P binding p roteins of known structure. GAD's cofactor binding site is particularl y interesting because GAD activity in the brain is controlled in part by a regulated interconversion of the apo-and holoenzymes. PROBE ident ified six motifs shared by the two GADs and four proteins of known str ucture: bacterial ornithine decarboxylase, dialkylglycine decarboxylas e, aspartate aminotransferase, and tyrosine phenol-lyase. Five of the motifs corresponded to the alpha/beta elements and loops that form mos t of the conserved fold of the pyridoxal-P binding cleft of the four e nzymes of known structure; the sixth motif corresponded to a helical e lement of the small domain that closes when the substrate binds. Eight residues that interact with pyridoxal-P and a ninth residue that lies at the interface of the large and small domains were also identified. Eleven additional conserved residues were identified and their functi ons were evaluated by examining the proteins of known structure. The k ey residues that interact directly with pyridoxal-P were identical in ornithine decarboxylase and the two GADs, thus allowing us to make a s pecific structural prediction of the cofactor binding site of GAD. The strong conservation of the cofactor binding site in GAD indicates tha t the highly regulated transition between apo-and holoGAD is accomplis hed by modifications in this basic fold rather than through a novel fo lding pattern.