ENZYMATIC O-SULFATION OF TYROSINE RESIDUES IN HIRUDINS BY SULFOTRANSFERASE FROM EUBACTERIUM A-44

The enzymic O-sulfation of Tyr residues in a recombinant hirudin varia nt-1 (rHV-1) and its analog in which Glu61 and Glu62 were replaced by Tyr, [E61Y, E62Y]rHV-1, was carried out by use of sulfotransferase iso lated from an anaerobic bacterium from the human intestine, Eubacteriu m A-44. Although rHV-1 was not sulfated by this enzyme, the sulfation of [E61Y, E62Y]rHV-1 was observed, and three kinds of sulfated analog, whose C-terminal six amino acid residues were -PYY(SO3H)YLQ, -PYYY(SO 3H)LQ, and PYY(SO3H)Y(SO3H)LQ, were obtained. Among the sulfated hirud in analogs tested here, the Tyr62 and Tyr63 bisulfated [E61Y, E62Y]rHV -1 showed the strongest thrombin inhibition with the inhibition consta nt (K-i) of 0.0430 pM, followed by the Tyr63 monosulfated analog (K-i = 0.0593 pM) and the Tyr62 monosulfated one (K-i = 0.158 pM). The Tyr6 3 monosulfated analog and Tyr62 and Tyr63 bisulfated one were more pot ent inhibitors of thrombin than unsulfated rHV-1. The increase in affi nity caused by sulfation was predominantly due to an increase in the a ssociation-rate constant.