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. Copyright © 1990, Wiley Blackwell. All rights reserved