Identification of Serine and Histidine as Essential Amino-Acid Residues in the Coagulant Enzyme Ancrod

1 Ancrod is inactivated by diisopropyl [32P]phosphofluoridate when one g-atom of 32P is irreversibly bound to 34600 g enzyme. 2 Acid hydrolysis of the radioactive and inactive enzyme confirms that the label is attached to a serine residue in l-serine-O-[32P]phosphate. 3 Ancrod is inactivated by the substrate analogue α-N-(nitrobenzyloxycarbonyl)-l-arginyl chloromethyl ketone. Kinetic analysis shows that the irreversible inactivation is preceded by a reversible binding of inactivator to the enzyme. This inactivation is prevented by the addition of α-N-benzoyl-l-arginine ethyl ester. 4 The rate of inactivation of ancrod by the active-site-directed reagent depends on the ionisation of a group with pK(app) of 7.4. This group is probably the histidine residue which is lost when the enzyme is inactivated by this compound. 5 A modified figure of approximately 38000 is suggested for the molecular weight of the enzyme which contains one active site.