PDBsum entry: 1f2o

Hydrolase

PDB id: 1f2o

Contents

Protein chain

277 a.a. *

Ligands

LEU

Metals

_ZN ×2

_CA

Waters ×203

* Residue conservation analysis

PDB id:

1f2o

Name:

Hydrolase

Title:

Crystal structure of the streptomyces griseus aminopeptidase complexed with l-leucine

Structure:

Aminopeptidase. Chain: a. Synonym: sgap. Ec: 3.4.11.-

Source:

Streptomyces griseus. Organism_taxid: 1911. Other_details: the enzyme is isolated from the commercially available enzyme mixture "pronase e"

Resolution:

1.70Å R-factor: 0.147 R-free: 0.175

Authors:

R.Gilboa,A.Spungin-Bialik,G.Wohlfahrt,D.Schomburg, S.Blumberg,G.Shoham

Key ref:

R.Gilboa et al. (2001). Interactions of Streptomyces griseus aminopeptidase with amino acid reaction products and their implications toward a catalytic mechanism. Proteins, 44, 490-504. PubMed id: 11484227 DOI: 10.1002/prot.1115

Date:

28-May-00 Release date: 22-Aug-01

Protein chain

P80561  (APX_STRGR) -  Aminopeptidase S

Seq: 284 a.a.

Struc: 277 a.a.

Enzyme reactions

• Enzyme class: E.C.3.4.11.24 - Aminopeptidase S.
• Cofactor: Zinc

 Gene Ontology (GO) functional annotation 

• Cellular component: extracellular region (1 term)
• Biological process: proteolysis (1 term)
• Biochemical function: hydrolase activity (4 terms)

DOI no: 10.1002/prot.1115

Proteins 44:490-504 (2001)

PubMed id: 11484227

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

R.Gilboa, A.Spungin-Bialik, G.Wohlfahrt, D.Schomburg, S.Blumberg, G.Shoham.

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.

Selected figure(s)

Figure 4.
Figure 4. a: Interactions of the bound leucine with SGAP as shown in a schematic diagram of the active site region of the SGAP/Leu complex. The protein bonds are shown in orange, the bound leucine (Leu) bonds are shown in purple, the Zn atoms are shown in green, whereas the rest of the atoms have the standard atomic colors. Dashed lines indicate hydrogen bonds or ionic interactions, whereas radiating spheres indicate hydrophobic contacts between the bound leucine (small spheres) and the neighboring protein groups (larger spheres). b: A similar diagram showing the interactions of the bound phenylalanine with SGAP in the SGAP/Phe complex. [Figs.

Figure 9.
Figure 9. Stereoview comparison of the active site of the MAP/MPA complex (2.0 Å structure, red; MPA in orange) with the active site of the LAP/LPA complex (1.65 Å structure, green; LPA in yellow).

The above figures are reprinted by permission from John Wiley & Sons, Inc.: Proteins (2001, 44, 490-504) copyright 2001.

Figures were selected by an automated process.