A kinetic study of the reaction between human myeloperoxidase, hydroperoxides and cyanide. Inhibition by chloride and thiocyanate.

The reaction between myeloperoxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7), hydrogen peroxide and ethyl hydroperoxide was investigated using the stopped-flow technique. Like other peroxidases, myeloperoxidase forms two sequential peroxide compounds. The pH-dependence of the apparent second-order rate constant of compound I formation shows that there is an acid/base group on the enzyme with a pKa of 4.30 +/- 0.15, which - when protonated - prevents the reaction of the enzyme with peroxides. The rate constants for the formation of compound I by hydrogen peroxide and ethyl hydroperoxide are (2.3 +/- 0.1) X 10(7) M-1 X s-1 and (2.8 +/- 0.3) X 10(5) M-1 X s-1, respectively. The binding of cyanide to myeloperoxidase (k1 = (1.30 +/- 0.05) X 10(6) M-1 X s-1) is also regulated by an acid/base group with a pKa of 4.00 +/- 0.05 as is the case with hydrogen peroxide; also, only the protonated uncharged form of cyanide reacts with the enzyme. From their effects on the binding of cyanide to the enzyme it is concluded that chloride and thiocyanate bind to myeloperoxidase only when the acid/base group is protonated. The pH-dependence of the dissociation constant of the myeloperoxidase-chloride complex obtained from the spectral changes induced by chloride is the same as observed in the inhibition by chloride of the binding of cyanide. It is concluded that hydrogen peroxide, cyanide, chloride and thiocyanate bind at the same site on the enzyme.