Insight into the coupling mechanism of the vitamin K-dependent carboxylase: mutation of histidine 160 disrupts glutamic acid carbanion formation and efficient coupling of vitamin K epoxidation to glutamic acid carboxylation.
Vitamin K-dependent (VKD) proteins become activated by the VKD carboxylase, which converts Glu's to carboxylated Glu's (Gla's) in their Gla domains. The carboxylase uses vitamin K epoxidation to drive Glu carboxylation, and the two half-reactions are coupled in 1:1 stoichiometry by an unknown mechanism. We now report the first identification of a residue, His160, required for coupling. A H160A mutant showed wild-type levels of epoxidation but substantially less carboxylation. Monitoring proton abstraction using a peptide with Glu tritiated at the gamma-carbon position revealed that poor coupling was due to impaired carbanion formation. H160A showed a 10-fold lower ratio of tritium release to vitamin K epoxidation than wild-type enzyme (i.e., 0.12 versus 1.14, respectively), which could fully account for the fold decrease in coupling efficiency. The Ala substitution in His160 did not affect the K m for vitamin K and caused only a 2-fold increase in the K m for Glu and 2-fold decrease in the activation of vitamin K epoxidation by Glu. The H160A K m for CO 2 was 5-fold higher than the wild-type enzyme. However, the k cat for H160A carboxylation was 8-9-fold lower than the wild-type enzyme with all three substrates (i.e., Glu, CO 2, and vitamin K), suggesting a catalytic role for His160 in carbanion formation. We propose that His160 facilitates the formation of the transition state for carbanion formation. His160 is highly conserved in metazoan VKD carboxylases but not in some bacterial orthologues (acquired by horizontal gene transfer), which has implications for how bacteria have adapted the carboxylase for novel functions.
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.
Berkner
Kathleen L
KL
D000838:Anions / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism; D019735:Carbon-Carbon Ligases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018698:Glutamic Acid / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D014812:Vitamin K / Q000737:chemistry / Q000378:metabolism*
D000838:Anions; D014812:Vitamin K; D002245:Carbon Dioxide; D018698:Glutamic Acid; D006639:Histidine; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase