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

FEBS Letters

Kivirikko, Kari I¹  Passoja, Kaisa¹  Pirskanen, Asta¹  Myllyharju, Johanna¹ 

[1] Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, Kajaanintie 52A, FIN-90220 Oulu, Finland

关键词: Lysyl hydroxylase;    2-Oxoglutarate;    Dioxygenase;    Catalytic site;    Collagen;   

DOI  :  10.1016/S0014-5793(98)00966-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 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.

【 授权许可】

Unknown