IDENTIFICATION OF THE 5 HYDROPHILIC RESIDUES (LYS-217, LYS-218, ARG-359, HIS-360, AND ARG-513) ESSENTIAL FOR THE STRUCTURE AND ACTIVITY OF VITAMIN-K-DEPENDENT CARBOXYLASE

Vitamin K-dependent carboxylase catalyzes the posttranslational conver sion of glutamic acid to gamma-carboxyglutamic acid in vitamin K-depen dent proteins. The clustered charged-to-alanine scanning mutagenesis o f bovine carboxylase has identified five distinct candidate regions (I . Sugiura et al., J. Biol. Chem. 271, 17837-17844, 1996) with signific ant loss-of-function phenotype. To further specify the residues essent ial for the structure and function of the enzyme, Lys-217, Lys-218, Ar g-359, His-360, Lys-361, Arg-513, and Lys-515 were analyzed by substit uting to alanine individually. All the mutants except for K217A were e xpressed in Chinese hamster ovary cells. The carboxylase activities of R359A, H360A, and R513A decreased in parallel with the vitamin K epox idase activities. Both carboxylations by R359A and H360A were stimulat ed saturatively at 1 mu M factor IX propeptide (pro-FIX18) concentrati on, but that by R513A was not at a concentration up to 128 mu M. K218A completely lost the enzyme activities but it cross-linked to the prop eptide, suggesting that Lys-218 is critical for enzyme activity withou t affecting propeptide binding. We conclude that Lys-218, Arg-359, and His-360 are involved in the catalytic event, and Arg-513 participates in propeptide binding. (C) 1998 Academic Press.