Structural evidence for leucine at the reactive site of heparin cofactor II.
The reaction products formed during the enzymatic inactivation of heparin cofactor II (HCII) by a proteinase isolated from Echis carinatus were analyzed by sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis and by reverse-phase high-performance liquid chromatography. By NaDodSO4-polyacrylamide gel electrophoresis, limited proteolysis of HCII was observed, which resulted in a decrease in the apparent molecular weight of the protein from approximately 68 000 to approximately 53 000. By reverse-phase high-performance liquid chromatography, at least 20 peptides were observed. Primary structure analysis of these peptides indicated that significant proteolysis had occurred in the NH2-terminal region of the protein. HCII inactivation, however, coincided with the appearance of a peptide from the COOH-terminal region of the protein. The peptide differed from the previously identified reactive site peptide [Griffith, M. J., Noyes, C. M., & Church, F. C. (1985) J. Biol. Chem. 260, 2218-2225] by only one residue: a leucyl residue at the NH2-terminal of the peptide. We conclude that leucine, as opposed to the expected arginine, is at the reactive site of HCII.
D000991:Antithrombins / Q000378:metabolism; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism; D015844:Heparin Cofactor II; D006801:Humans; D007700:Kinetics; D007930:Leucine; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010447:Peptide Hydrolases / Q000378:metabolism
D000991:Antithrombins; D006023:Glycoproteins; D010446:Peptide Fragments; D015844:Heparin Cofactor II; D010447:Peptide Hydrolases; D007930:Leucine