Aminopeptidase A (EC 3.4.11.7; APA) is a 130 kDa membrane-bound zinc enzyme that contains the consensus sequence HEXXH (residues 385-389) conserved among the zinc metalloprotease family. In this motif, both histidine residues and the glutamic residue were shown to be involved respectively in zinc co-ordination and catalytic activity. Treatment of APA with N-acetylimidazole results in a loss of enzymic activity; this is prevented by the competitive aminopeptidase inhibitor amastatin, suggesting the presence of an important tyrosine, lysine or cysteine residue at the active site of APA. A tyrosine residue was previously proposed to be involved in the enzymic activity of aminopeptidase N. Furthermore sequence alignment of mouse APA with other monozinc aminopeptidases indicates the presence of a conserved tyrosine (Tyr-471 in APA). The functional role of Tyr-471 in APA was investigated by replacing this residue with a phenylalanine (Phe-471) or a histidine (His-471) residue by site-directed mutagenesis. Kinetic studies showed that the Km values of both mutants were similar to that of the wild-type enzyme, whereas kcat values were decreased by three orders of magnitude and corresponded to a variation in free energy of the rate-limiting step by 4.0 and 4.2 kcal/mol (0.96 and 1.00 kJ/mol) for the Phe-471 and His-471 mutants respectively. The mutation did not modify the inhibitory potency of a thiol-containing inhibitor that strongly chelates the active-site zinc ion, whereas that of a putative analogue of the transition state presumed to mimic the reaction intermediate was reduced. Taken together, these results strongly suggest that the Tyr-471 hydroxy group participates in catalysis by stabilizing the transition state complex through interaction with the oxyanion.
Skip Nav Destination
Article navigation
November 1997
-
Cover Image
Cover Image
- PDF Icon PDF LinkFront Matter
- PDF Icon PDF LinkTable of Contents
Research Article|
November 01 1997
A tyrosine residue essential for catalytic activity in aminopeptidase A
Gilles VAZEUX;
Gilles VAZEUX
1INSERM Unit 36, Collège de France, 3 rue d'Ulm, 75005 Paris, France
Search for other works by this author on:
Xavier ITURRIOZ;
Xavier ITURRIOZ
1INSERM Unit 36, Collège de France, 3 rue d'Ulm, 75005 Paris, France
Search for other works by this author on:
Pierre CORVOL;
Pierre CORVOL
1INSERM Unit 36, Collège de France, 3 rue d'Ulm, 75005 Paris, France
Search for other works by this author on:
Catherine LLORENS-CORTÈS
Catherine LLORENS-CORTÈS
1
1INSERM Unit 36, Collège de France, 3 rue d'Ulm, 75005 Paris, France
1To whom correspondence should be addressed.
Search for other works by this author on:
Publisher: Portland Press Ltd
Accepted:
July 07 1996
Received:
April 02 1997
Revision Received:
June 19 1997
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1997
1997
Biochem J (1997) 327 (3): 883–889.
Article history
Accepted:
July 07 1996
Received:
April 02 1997
Revision Received:
June 19 1997
Citation
Gilles VAZEUX, Xavier ITURRIOZ, Pierre CORVOL, Catherine LLORENS-CORTÈS; A tyrosine residue essential for catalytic activity in aminopeptidase A. Biochem J 1 November 1997; 327 (3): 883–889. doi: https://doi.org/10.1042/bj3270883
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Captcha Validation Error. Please try again.