Identification of arginine-700 as the residue that binds the C-5 carboxyl group of 2-oxoglutarate in human lysyl hydroxylase 1

• Published in: FEBS letters Vol. 434; no. 1; pp. 145 - 148
• Main Authors: Passoja, Kaisa, Myllyharju, Johanna, Pirskanen, Asta, Kivirikko, Kari I
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 28-08-1998
• Subjects: 2-Oxoglutarate; Amino Acid Sequence; Arginine; Binding Sites; Carboxylic Acids; Catalytic site; Collagen; Dioxygenase; Humans; Ketoglutaric Acids - metabolism; Lysyl hydroxylase; Molecular Sequence Data; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase - genetics; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase - metabolism; Dioxygenase; Catalytic site; Lysyl hydroxylase; 2-Oxoglutarate; Collagen
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(98)00966-1

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 Fe
2+ 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
K
m for 2-oxoglutarate, whereas the
V
max 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.