title: Identification of arginine-700 as the residue that binds the C-5 carboxyl group of 2-oxoglutarate in human lysyl hydroxylase 1.
abstract: Lysyl hydroxylase catalyzes the formation of hydroxylysine in collagens by a reaction that involves oxidative decarboxylation of 2-oxoglutarate. Its binding site can be divided into two main subsites: subsite I consists of a positively charged side-chain which binds the C-5 carboxyl group, while subsite II consists of two coordination sites of the enzyme-bound Fe2+ and is chelated by the C-1-C-2 moiety. In order to identify subsite I, we converted Arg-697, Arg-700 and Ser-705 individually to alanine and Arg-700 also to lysine, and expressed the mutant enzymes in insect cells. Arg-700-Ala inactivated lysyl hydroxylase completely, whereas Arg-697-Ala and Ser-723-Ala had only a relatively minor effect. Arg-700-Lys produced 93% inactivation under standard assay conditions, the main effect being a 10-fold increase in the Km for 2-oxoglutarate, whereas the Vmax was unchanged. Arg-700 thus provides the positively charged residue that binds the C-5 carboxyl group of 2-oxoglutarate, whereas Ser-705 appears to be of no functional significance in this binding.
authors.affiliation: Biocenter and Department of Medical Biochemistry, University of Oulu, Finland.
authors.forename: K
authors.initials: K
authors.lastname: Passoja
authors.forename: J
authors.initials: J
authors.lastname: Myllyharju
authors.forename: A
authors.initials: A
authors.lastname: Pirskanen
authors.forename: K I
authors.initials: KI
authors.lastname: Kivirikko
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine; D001665:Binding Sites; D002264:Carboxylic Acids; D006801:Humans; D007656:Ketoglutaric Acids / Q000378:metabolism*; D008969:Molecular Sequence Data; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase / Q000235:genetics / Q000378:metabolism*
chemical_list: D002264:Carboxylic Acids; D007656:Ketoglutaric Acids; D001120:Arginine; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase