FURTHER-STUDIES ON THE ENZYMATIC CONVERSION OF ACETYL-COENZYME-A INTO3-HYDROXY-3-METHYLGLUTARYL-COENZYME-A IN RADISH

We purified from radish (Raphanus sativus L.) membranes (P 16,000 x g, isolated from 4-day-old etiolated seedlings) an enzyme system capable of catalyzing the two-step conversion of acetyl-CoA into 3-hydroxy-3- methylglutaryl(HMG)-CoA. Since it is known for yeast and mammalian tis sues that two enzyme activities are involved in HMG-CoA synthesis (ace toacetyl-CoA thiolase, AACT, EC 2.1.3.9, and HMG-CoA synthase, HMGS, E C 4.1.3.5), this enzyme system is further referred to as AACT/HMGS. Ap parent AACT/HMGS activity is high in solubilized membranes and gets in creasingly lost during the purification. Its activity can be reconstit uted by addition of boiled solubilisate, or by addition of Fe2(+)-chel ates and of quinone cofactors, with pyrroloquinoline quinone (PQQ) bei ng the most effective one studied so far. Gel filtration experiments o f enzyme after preincubation in presence of pQQ indicate that there is no covalent binding of the quinone cofactor to the enzyme. We suggest a reaction mechanism that involves radical formation by the redox cou ple Fe2+/PQQ which seems to mimick the natural cofactor system yet to be identified. The quinone cofactor can be partially replaced by high concentrations of H2O2, possibly indicative of hydroxyl radical format ion to be essential for the apparent enzyme activation.