Initiation of chondroitin sulfate biosynthesis: a kinetic analysis of UDP-D-xylose: core protein beta-D-xylosyltransferase.
The nature of the primary signals important for the addition of xylose to serines on the core protein of the cartilage chondroitin sulfate proteoglycan has been investigated. The importance of consensus sequence elements (Acidic-Acidic-Xxx-Ser-Gly-Xxx-Gly) in the natural acceptor was shown by the significant decrease in acceptor capability of peptide fragments derived by digestion of deglycosylated core protein with Staphylococcus aureus V8 protease, which cleaves within the consensus sequence, compared to the similar reactivity of trypsin-derived peptide fragments, in which consensus sequences remain intact. A comparison of the acceptor efficiencies (Vmax/Km) of synthetic peptides containing the proposed xylosylation consensus sequence and the natural acceptor (deglycosylated core protein) was then made by use of the in vitro xylosyltransferase assay. The two types of substrates were found to have nearly equivalent acceptor efficiencies and to be competitive inhibitors of each other's acceptor capability, with Km = Kiapparent. These results suggest that the artificial peptides containing the consensus sequence are analogues of individual substitution sites on the core protein and allowed the kinetic mechanism of the xylosyltransferase reaction to be investigated, with one of the artificial peptides as a model substrate. The most probable kinetic mechanism for the xylosyltransferase reaction was found to be an ordered single displacement with UDP-xylose as the leading substrate and the xylosylated peptide as the first product released. This represents the first reported formal kinetic mechanism for this glycosyltransferase and the only one reported for a nucleotide sugar:protein transferase.
Kearns
A E
AE
Department of Pediatrics, University of Chicago, Illinois 60637.
Campbell
S C
SC
Westley
J
J
Schwartz
N B
NB
D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D011508:Chondroitin Sulfate Proteoglycans / Q000096:biosynthesis / Q000378:metabolism*; D002813:Chondrosarcoma / Q000201:enzymology; D010450:Endopeptidases / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010430:Pentosyltransferases / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D051381:Rats; D012694:Serine / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014530:Uridine Diphosphate / Q000378:metabolism; D014540:Uridine Diphosphate Xylose / Q000378:metabolism; D014994:Xylose / Q000378:metabolism*; D000097608:UDP Xylose-Protein Xylosyltransferase
D011508:Chondroitin Sulfate Proteoglycans; D010450:Endopeptidases; D010430:Pentosyltransferases; D010446:Peptide Fragments; D012694:Serine; D014357:Trypsin; D014530:Uridine Diphosphate; D014540:Uridine Diphosphate Xylose; D014994:Xylose; D000097608:UDP Xylose-Protein Xylosyltransferase