SITE-DIRECTED MUTAGENESIS OF HUMAN LYSYL HYDROXYLASE EXPRESSED IN INSECT CELLS - IDENTIFICATION OF HISTIDINE-RESIDUES AND AN ASPARTIC-ACID RESIDUE CRITICAL FOR CATALYTIC ACTIVITY

Citation
A. Pirskanen et al., SITE-DIRECTED MUTAGENESIS OF HUMAN LYSYL HYDROXYLASE EXPRESSED IN INSECT CELLS - IDENTIFICATION OF HISTIDINE-RESIDUES AND AN ASPARTIC-ACID RESIDUE CRITICAL FOR CATALYTIC ACTIVITY, The Journal of biological chemistry, 271(16), 1996, pp. 9398-9402
Citations number
31
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology
ISSN journal
0021-9258
Volume
271
Issue
16
Year of publication
1996
Pages
9398 - 9402
Database
ISI
SICI code
0021-9258(1996)271:16<9398:SMOHLH>2.0.ZU;2-R
Abstract
Lysyl hydroxylase (EC 1.14.11.4), an alpha(2) homodimer, catalyzes the formation of hydroxylysine in collagens. We expressed here human lysy l hydroxylase in insect cells by baculovirus vectors, About 90% of the enzyme produced was soluble 32 h after infection, whereas only 10% wa s soluble at 72 h, Twelve histidines, five aspartates, and all four as paragines that may act as N-glycosylation sites were converted individ ually to serine, alanine, or glutamine, respectively, and the mutant e nzymes were expressed in insect cells, Three histidine mutations and o ne aspartate mutation appeared to inactivate the enzyme completely, Th ese and other data suggest that histidines 656 and 708 and aspartate 6 58 provide the three ligands required for the binding of Fe2+ to a cat alytic site, whereas the role of the third critical histidine (residue 706) remains to be established, Three additional histidine mutations also had a major effect, although they did not inactivate the enzyme c ompletely, whereas six further histidine mutations and four out of fiv e aspartate mutations had a much more minor effect. Data on the four a sparagine mutations suggested that only two of the potential N-glycosy lation sites may be fully glycosylated in insect cells and that one of these carbohydrate units may be needed for full enzyme activity.