IDENTIFICATION OF ARGININE-700 AS THE RESIDUE THAT BINDS THE C-5 CARBOXYL GROUP OF 2-OXOGLUTARATE IN HUMAN LYSYL HYDROXYLASE-1

Citation
K. Passoja et al., IDENTIFICATION OF ARGININE-700 AS THE RESIDUE THAT BINDS THE C-5 CARBOXYL GROUP OF 2-OXOGLUTARATE IN HUMAN LYSYL HYDROXYLASE-1, FEBS letters, 434(1-2), 1998, pp. 145-148
Citations number
34
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology,"Cell Biology",Biophysics
Journal title
ISSN journal
0014-5793
Volume
434
Issue
1-2
Year of publication
1998
Pages
145 - 148
Database
ISI
SICI code
0014-5793(1998)434:1-2<145:IOAATR>2.0.ZU;2-R
Abstract
Lysyl hydroxylase catalyzes the formation of hydroxylysine in collagen s by a reaction that involves oxidative decarboxylation of 2-oxoglutar ate. Its binding site can be divided into two main subsites: subsite I consists of a positively charged side-chain which binds the C-5 carbo xyl group, while subsite Il consists of two coordination sites of the enzyme-bound Fe2+ and is chelated by the C-1-C-2 moiety, In order to i dentify subsite I, we converted Arg-697, Arg-700 and Ser-705 individua lly to alanine and Arg-700 also to lysine, and expressed the mutant en zymes in insect cells. Arg-700-Ala inactivated lysyl hydroxylase compl etely, whereas Arg-697-Ala and Ser-723-Ala had only a relatively minor effect. Arg-700-Lys produced 93% inactivation under standard assay co nditions, the main effect being a 10-fold increase in the K-m for 2-ox oglutarate, whereas the V-max was unchanged. Arg-700 thus provides the positively charged residue that binds the C-5 carboxyl group of 2-oxo glutarate, whereas Ser-705 appears to be of no functional significance in this binding. (C) 1998 Federation of European Biochemical Societie s.