Brønsted analysis reveals Lys218 as the carboxylase active site base that deprotonates vitamin K hydroquinone to initiate vitamin K-dependent protein carboxylation.
The vitamin K-dependent (VKD) carboxylase converts Glu's to carboxylated Glu's in VKD proteins to render them functional in a broad range of physiologies. The carboxylase uses vitamin K hydroquinone (KH(2)) epoxidation to drive Glu carboxylation, and one of its critical roles is to provide a catalytic base that deprotonates KH(2) to allow epoxidation. A long-standing model invoked Cys as the catalytic base but was ruled out by activity retention in a mutant where every Cys is substituted by Ala. Inhibitor analysis of the cysteine-less mutant suggested that the base is an activated amine [Rishavy et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 13732-13737], and in the present study, we used an evolutionary approach to identify candidate amines, which revealed His160, His287, His381, and Lys218. When mutational analysis was performed using an expression system lacking endogenous carboxylase, the His to Ala mutants all showed full epoxidase activity but K218A activity was not detectable. The addition of exogenous amines restored K218A activity while having little effect on wild type carboxylase, and pH studies indicated that rescue was dependent upon the basic form of the amine. Importantly, Brønsted analysis that measured the effect of amines with different pK(a) values showed that K218A activity rescue depended upon the basicity of the amine. The combined results provide strong evidence that Lys218 is the essential base that deprotonates KH(2) to initiate the reaction. The identification of this base is an important advance in defining the carboxylase active site and has implications regarding carboxylase membrane topology and the feedback mechanism by which the Glu substrate regulates KH(2) oxygenation.
Rishavy
Mark A
MA
Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic Lerner College of Medicine at Case Western Reserve University, 9500 Euclid Avenue, Cleveland, Ohio 44195, USA.
Hallgren
Kevin W
KW
Yakubenko
Anna V
AV
Shtofman
Rebecca L
RL
Runge
Kurt W
KW
Berkner
Kathleen L
KL
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019735:Carbon-Carbon Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002264:Carboxylic Acids / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017386:Sequence Homology, Amino Acid; D024482:Vitamin K 2 / Q000378:metabolism*
D002264:Carboxylic Acids; D024482:Vitamin K 2; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase; D008239:Lysine