2ANP

Functional Glutamate 151 to Histidine mutant of the aminopeptidase from Aeromonas Proteolytica.

Summary for 2ANP

• Entry DOI: 10.2210/pdb2anp/pdb
• Related: 1AMP 1CP6 1LOK 1TXR
• Descriptor: leucyl aminopeptidase, ZINC ION, SODIUM ION, ... (4 entities in total)
• Functional Keywords: aminopeptidase, bi-metallic, zinc, epr, spectroscopy, hydrolase
• Biological source: Vibrio proteolyticus
• Cellular location: Secreted : Q01693
• Total number of polymer chains: 1
• Total formula weight: 31590.19

Authors

Bzymek, K.P.,Moulin, A.,Swierczek, S.I.,Ringe, D.,Petsko, G.A.,Holz, R.C. (deposition date: 2005-08-11, release date: 2005-10-04, Last modification date: 2024-10-09)

Primary citation

Bzymek, K.P.,Moulin, A.,Swierczek, S.I.,Ringe, D.,Petsko, G.A.,Bennett, B.,Holz, R.C.
Kinetic, Spectroscopic, and X-ray Crystallographic Characterization of the Functional E151H Aminopeptidase from Aeromonas proteolytica.
Biochemistry, 44:12030-12040, 2005

PubMed Abstract: Glutamate151 (E151) has been shown to be catalytically essential for the aminopeptidase from Vibrio proteolyticus (AAP). E151 acts as the general acid/base during the catalytic mechanism of peptide hydrolysis. However, a glutamate residue is not the only residue capable of functioning as a general acid/base during catalysis for dinuclear metallohydrolases. Recent crystallographic characterization of the D-aminopeptidase from Bacillus subtilis (DppA) revealed a histidine residue that resides in an identical position to E151 in AAP. Because the active-site ligands for DppA and AAP are identical, AAP has been used as a model enzyme to understand the mechanistic role of H115 in DppA. Substitution of E151 with histidine resulted in an active AAP enzyme exhibiting a kcat value of 2.0 min(-1), which is over 2000 times slower than r AAP (4380 min(-1)). ITC experiments revealed that ZnII binds 330 and 3 times more weakly to E151H-AAP compared to r-AAP. UV-vis and EPR spectra of CoII-loaded E151H-AAP indicated that the first metal ion resides in a hexacoordinate/pentacoordinate equilibrium environment, whereas the second metal ion is six-coordinate. pH dependence of the kinetic parameters kcat and K(m) for the hydrolysis of L-leucine p-nitroanilide (L-pNA) revealed a change in an ionization constant in the enzyme-substrate complex from 5.3 in r-AAP to 6.4 in E151H-AAP, consistent with E151 in AAP being the active-site general acid/base. Proton inventory studies at pH 8.50 indicate the transfer of one proton in the rate-limiting step of the reaction. Moreover, the X-ray crystal structure of [ZnZn(E151H-AAP)] has been solved to 1.9 A resolution, and alteration of E151 to histidine does not introduce any major conformational changes to the overall protein structure or the dinuclear ZnII active site. Therefore, a histidine residue can function as the general acid/base in hydrolysis reactions of peptides and, through analogy of the role of E151 in AAP, H115 in DppA likely shuttles a proton to the leaving group of the substrate.

PubMed: 16142900
DOI: 10.1021/bi0505823

Experimental method

X-RAY DIFFRACTION (1.9 Å)