Characteristics and composition of the vitamin K-dependent gamma-glutamyl carboxylase-binding domain on osteocalcin.
Two different sites on vitamin K-dependent gamma-glutamyl carboxylase (VKC) are involved in enzyme-substrate interaction: the propeptide-binding site required for high-affinity substrate binding and the active site for glutamate carboxylation. Synthetic descarboxy osteocalcin (d-OC) is a low-K(m) substrate for the VKC, but unique since it possesses a high-affinity recognition site for the VKC, distinct from the propeptide which is essential as a binding site for VKC. However, the exact location and composition of this VKC-recognition domain on d-OC has remained unclear until now. Using a stereospecific substrate analogue [t-butyloxycarbonyl-(2S,4S)-4-methylglutamic acid-Glu-Val (S-MeTPT)] we demonstrate in this paper that the high affinity of d-OC for VKC cannot be explained by a direct interaction with either the active site or with the propeptide-binding site on VKC. It is shown using various synthetic peptides derived from d-OC that there are two domains on d-OC necessary for recognition: one located between residues 1 and 12 and a second between residues 26 and 39, i.e. at the C-terminal side of the gamma-carboxyglutamate (Gla) domain. Both internal sequences contribute substantially to the efficiency of carboxylation. On the basis of these data we postulate the presence of a second high-affinity substrate-binding site on VKC capable of specifically binding d-OC, which is the first vitamin K-dependent substrate of which the VKC binding domain is interrupted by the Gla domain.
Houben
Roger J T J
RJ
Department of Biochemistry and Cardiovascular Research Institute, Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands.
Rijkers
Dirk T S
DT
Stanley
Thomas B
TB
Acher
Francine
F
Azerad
Robert
R
Käkönen
Sanna-Maria
SM
Vermeer
Cees
C
Soute
Berry A M
BA
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019735:Carbon-Carbon Ligases / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D007313:Insecta; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D015675:Osteocalcin / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014812:Vitamin K / Q000378:metabolism*
D015675:Osteocalcin; D014812:Vitamin K; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase