Interactions of Streptomyces griseus aminopeptidase with amino acid reaction products and their implications toward a catalytic mechanism

Streptomyces griseus aminopeptidase (SGAP) is a double-zinc exopeptidase wi th a high preference toward large hydrophobic amino-terminus residues. It i s a monomer of a relatively low molecular weight (30 kDa), it is heat stabl e, it displays a high and efficient catalytic turnover, and its activity is modulated by calcium ions. The small size, high activity, and heat stabili ty make SGAP a very attractive enzyme for various biotechnological applicat ions, among which is the processing of recombinant DNA proteins and fusion protein products. Several free amino acids, such as phenylalanine, leucine, and methionine, were found to act as weak inhibitors of SGAP and hence wer e chosen for structural studies. These inhibitors can potentially be regard ed as product analogs because one of the products obtained in a normal enzy matic reaction is the cleaved amino terminal amino acid of the substrate. T he current study includes the X-ray crystallographic analysis of the SGAP c omplexes with methionine (1.53 Angstrom resolution), leucine (1.70 Angstrom resolution), and phenylalanine (1.80 Angstrom resolution). These three hig h-resolution structures have been used to fully characterize the SGAP activ e site and to identify some of the functional groups of the enzyme that are involved in enzyme-substrate and enzyme-product interactions. A unique bin ding site for the terminal amine group of the substrate (including the side chains of Glu131 and Asp160, as well as the carbonyl group of Arg202) is i ndicated to play an important role in the binding and orientation of both t he substrate and the product of the catalytic reaction. These studies also suggest that Glu131 and Tyr246 are directly involved in the catalytic mecha nism of the enzyme. Both of these residues seem to be important for substra te binding and orientation, as well as the stabilization of the tetrahedral transition state of the enzyme-substrate complex. Glu131 is specifically s uggested to function as a general base during catalysis by promoting the nu cleophilic attack of the zinc-bound water/hydroxide on the substrate carbon yl carbon. The structures of the three SGAP complexes are compared with rec ent structures of three related aminopeptidases: Aeromonas proteolytica ami nopeptidase (AAP), leucine aminopeptidase (LAP), and methionine aminopeptid ase (MAP) and their complexes with corresponding inhibitors and analogs. Th ese structural results have been used for the simulation of several species along the reaction coordinate and for the suggestion of a general scheme f or the proteolytic reaction catalyzed by SGAP. Proteins 2001;44:490-504. (C ) 2001 Wiley-Liss, Inc.