The effect of substituting phosphotyrosine for sulphotyrosine on the activity of hirudin.
Recombinant hirudin (hirudin), which lacks the sulphate group on Tyr-63, has a tenfold-reduced affinity for alpha-thrombin. Incubation of recombinant hirudin with [gamma-32P]ATP and protein tyrosine kinase III from spleen resulted in incorporation of radioactivity into the protein. Phosphatohirudin was purified to homogeneity (overall yield 5%) and shown to contain 1 mol phosphate/mol protein, as a phosphotyrosyl residue at position 63. The kinetics of the inhibition of human alpha-thrombin by phosphatohirudin were determined. It was found that the introduction of the negatively charged phosphate had fully restored the affinity of recombinant hirudin for alpha-thrombin to the level of the wild-type sulphatohirudin. The inhibition constant of phosphatohirudin was 18 fM compared with 20 fM for that of sulphatohirudin. Moreover, the values for the on- and off-rate constants of both forms of hirudin were indistinguishable.
Friedrich Miescher Institut, Basel, Switzerland.
J
J
Hofsteenge
S R
SR
Stone
A
A
Donella-Deana
L A
LA
Pinna
D000255:Adenosine Triphosphate / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites / Q000187:drug effects; D002852:Chromatography, Ion Exchange; D006629:Hirudins / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D010449:Peptide Mapping; D010766:Phosphorylation; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D013917:Thrombin / Q000037:antagonists & inhibitors*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000096:biosynthesis
D000596:Amino Acids; D006629:Hirudins; D011994:Recombinant Proteins; D019000:Phosphotyrosine; C005662:tyrosine O-sulfate; D014443:Tyrosine; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D013917:Thrombin