Osteocalcin binds tightly to the gamma-glutamylcarboxylase at a site distinct from that of the other known vitamin K-dependent proteins.
Vitamin K-dependent proteins contain a propeptide that is required for recognition by the enzyme gamma-glutamylcarboxylase. Substrates used in vitro for carboxylation studies lacking a prosequence are characterized by Km values in the millimolar range, whereas the Km for peptides containing a prosequence is three or four orders of magnitude smaller. Here we report that descarboxy-osteocalcin is an exception in this respect. With descarboxy-osteocalcin in purified propeptide-free recombinant carboxylase, the Km was 1.8 microM. Furthermore, osteocalcin was an inhibitor of descarboxy-osteocalcin carboxylation with a Ki of 76 microM. In contrast with the other vitamin K-dependent proteins, free propeptides do not inhibit descarboxy-osteocalcin carboxylation. Moreover, propeptide-containing substrates were inhibited neither by osteocalcin nor by its propeptide. From our studies we conclude that descarboxy-osteocalcin must have an internal recognition sequence that binds to gamma-glutamylcarboxylase at a site different from the propeptide-recognition site.
Houben
R J
RJ
Department of Biochemistry and Cardiovascular Research Institute, Maastricht University, P.O. Box 616, 6200 MD, Maastricht, The Netherlands.
Jin
D
D
Stafford
D W
DW
Proost
P
P
Ebberink
R H
RH
Vermeer
C
C
Soute
B A
BA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019735:Carbon-Carbon Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D008969:Molecular Sequence Data; D015675:Osteocalcin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D013379:Substrate Specificity; D014812:Vitamin K / Q000378:metabolism*
D010455:Peptides; D015675:Osteocalcin; D014812:Vitamin K; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase