title: Interactions of Streptomyces griseus aminopeptidase with amino acid reaction products and their implications toward a catalytic mechanism.
abstract: Streptomyces griseus aminopeptidase (SGAP) is a double-zinc exopeptidase with a high preference toward large hydrophobic amino-terminus residues. It is a monomer of a relatively low molecular weight (30 kDa), it is heat stable, it displays a high and efficient catalytic turnover, and its activity is modulated by calcium ions. The small size, high activity, and heat stability make SGAP a very attractive enzyme for various biotechnological applications, 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 were chosen for structural studies. These inhibitors can potentially be regarded as product analogs because one of the products obtained in a normal enzymatic reaction is the cleaved amino terminal amino acid of the substrate. The current study includes the X-ray crystallographic analysis of the SGAP complexes with methionine (1.53 A resolution), leucine (1.70 A resolution), and phenylalanine (1.80 A resolution). These three high-resolution structures have been used to fully characterize the SGAP active site and to identify some of the functional groups of the enzyme that are involved in enzyme-substrate and enzyme-product interactions. A unique binding 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 indicated to play an important role in the binding and orientation of both the substrate and the product of the catalytic reaction. These studies also suggest that Glu131 and Tyr246 are directly involved in the catalytic mechanism of the enzyme. Both of these residues seem to be important for substrate binding and orientation, as well as the stabilization of the tetrahedral transition state of the enzyme-substrate complex. Glu131 is specifically suggested to function as a general base during catalysis by promoting the nucleophilic attack of the zinc-bound water/hydroxide on the substrate carbonyl carbon. The structures of the three SGAP complexes are compared with recent structures of three related aminopeptidases: Aeromonas proteolytica aminopeptidase (AAP), leucine aminopeptidase (LAP), and methionine aminopeptidase (MAP) and their complexes with corresponding inhibitors and analogs. These structural results have been used for the simulation of several species along the reaction coordinate and for the suggestion of a general scheme for the proteolytic reaction catalyzed by SGAP.
authors.affiliation: Department of Inorganic Chemistry and the Laboratory for Structural Chemistry and Biology, The Hebrew University of Jerusalem, Jerusalem, Israel.
authors.forename: R
authors.initials: R
authors.lastname: Gilboa
authors.forename: A
authors.initials: A
authors.lastname: Spungin-Bialik
authors.forename: G
authors.initials: G
authors.lastname: Wohlfahrt
authors.forename: D
authors.initials: D
authors.lastname: Schomburg
authors.forename: S
authors.initials: S
authors.lastname: Blumberg
authors.forename: G
authors.initials: G
authors.lastname: Shoham
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D000626:Aminopeptidases / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D007931:Leucyl Aminopeptidase / Q000737:chemistry / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D063208:Methionyl Aminopeptidases; D008956:Models, Chemical; D008958:Models, Molecular; D010649:Phenylalanine / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D055672:Static Electricity; D013305:Streptomyces griseus / Q000201:enzymology*; D015032:Zinc / Q000378:metabolism
chemical_list: D000596:Amino Acids; D010649:Phenylalanine; D008715:Methionine; D000626:Aminopeptidases; D007931:Leucyl Aminopeptidase; D063208:Methionyl Aminopeptidases; D007930:Leucine; D015032:Zinc