The effect of substituting phosphotyrosine for sulphotyrosine on the activity of hirudin

Recombinant hirudin (hirudin), which lacks the sulphate group of Typ-63, has a tenfold-reduced affinity for α-thrombin. Incubation of recombinant hirudin with [γ-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 α-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 α-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 the both forms of hirudin were indistinguishable.