Role of the propeptide and gamma-glutamic acid domain of factor IX for in vitro carboxylation by the vitamin K-dependent carboxylase.
The vitamin K-dependent gamma-glutamyl carboxylase catalyzes the processive carboxylation of specific glutamates in a number of proteins related to blood coagulation and bone. To address the independent importance of the propeptide, gamma-carboxyglutamic acid (Gla) domain and elements beyond the Gla domain of factor IX in vitamin K-dependent carboxylation, we have examined the kinetics of carboxylation of peptides containing (1) propeptide and Gla domain, (2) the Gla domain alone, (3) uncarboxylated bone Gla protein, (4) propeptide followed by the entire uncarboxylated factor IX molecule, and (5) the factor IX propeptide followed by a non-Gla domain sequence. Our studies indicate that peptides with a covalently linked propeptide have Km values similar to the physiological substrate of the carboxylase. In contrast, the Gla domain of factor IX has a >/=230-fold higher Km for the carboxylase than the corresponding peptide with a covalently linked propeptide. This contrasts with bone Gla protein, another vitamin K-dependent protein, which appears not to require a covalently linked propeptide for high-affinity binding to the carboxylase. Analysis of the carboxylation products of a propeptide/non-Gla domain substrate indicate that it is carboxylated multiple times in a processive manner. These studies show that the perceived binding affinity of the carboxylase substrate and processivity is conferred by the propeptide without requiring the conserved Gla domain sequences and that factor IX and bone Gla protein may have distinct mechanisms of interacting with the carboxylase.
Stanley
T B
TB
Department of Biology, Center for Thrombosis and Hemostasis, University of North Carolina at Chapel Hill 27599-3280, USA.
Wu
S M
SM
Houben
R J
RJ
Mutucumarana
V P
VP
Stafford
D W
DW
D015055:1-Carboxyglutamic Acid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D019735:Carbon-Carbon Ligases / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D006224:Cricetinae; D005164:Factor IX / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D014812:Vitamin K / Q000737:chemistry
D010455:Peptides; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D014812:Vitamin K; D015055:1-Carboxyglutamic Acid; D005164:Factor IX; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase