PDBsum entry 2anp

Hydrolase

PDB id: 2anp

Contents

Protein chain

291 a.a. *

Metals

_NA

_ZN ×2

Waters ×136

* Residue conservation analysis

PDB id:

2anp

Name:

Hydrolase

Title:

Functional glutamate 151 to histidine mutant of the aminopeptidase from aeromonas proteolytica.

Structure:

Leucyl aminopeptidase. Chain: a. Engineered: yes. Mutation: yes

Source:

Vibrio proteolyticus. Organism_taxid: 671. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Monomer (from PQS)

Resolution:

1.90Å R-factor: 0.180 R-free: 0.205

Authors:

K.P.Bzymek,A.Moulin,S.I.Swierczek,D.Ringe,G.A.Petsko,R.C.Holz

Key ref:

K.P.Bzymek et al. (2005). Kinetic, spectroscopic, and X-ray crystallographic characterization of the functional E151H aminopeptidase from Aeromonas proteolytica. Biochemistry, 44, 12030-12040. PubMed id: 16142900 DOI: 10.1021/bi0505823

Date:

11-Aug-05 Release date: 04-Oct-05

Protein chain

Q01693  (AMPX_VIBPR) -  Bacterial leucyl aminopeptidase from Vibrio proteolyticus

Seq: 504 a.a.

Struc: 291 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.4.11.10 - bacterial leucyl aminopeptidase.
• Reaction: Release of an N-terminal amino acid, preferentially leucine, but not glutamic or aspartic acids.
• Cofactor: Zn(2+)

DOI no: 10.1021/bi0505823

Biochemistry 44:12030-12040 (2005)

PubMed id: 16142900

Kinetic, spectroscopic, and X-ray crystallographic characterization of the functional E151H aminopeptidase from Aeromonas proteolytica.

K.P.Bzymek, A.Moulin, S.I.Swierczek, D.Ringe, G.A.Petsko, B.Bennett, R.C.Holz.

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.