The time-dependent inactivation of human brain dihydropteridine reductase by the oxidation products of L-dopa.

Abstract

Dihydropteridine reductase (DHPR) was irreversibly inactivated in a time-dependent way by incubation with 3,4-dihydroxyphenylalanine (L-dopa). The inactivation was oxygen-dependent; incubation under nitrogen gave partial protection. The inactivation was stimulated by the presence of horse-radish peroxidase/hydrogen peroxide. L-Dopa itself was not an inhibitor of DHPR although dopachrome, the aminochrome formed following oxidation of L-dopa, was a reversible inhibitor of DHPR with an I50 of 0.60 mM. The quinone products of oxidation of L-dopa were responsible for the time-dependent inactivation of DHPR. Adrenochrome also demonstrated a time-dependent inactivation of DHPR. Inactivation by adrenochrome demonstrated a saturation effect suggesting the reversible formation of a complex preceding inactivation. No radiolabel was incorporated into DHPR following inactivation by L-[14C]-dopa. Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated the presence of a dimer of DHPR. A mechanism of inactivation involving the oxidative coupling of essential thiol groups was proposed to explain inactivation.