Human liver prenyltransferase and its characterization

Prenyltransferase (dimethylallydiphosphate : isopentenyldiphosphate dimethylallytransferase, EC 2.5.1.1) has been purified to homogeneity from human liver obtained at autopsy. The enzyme is a dimer with a native molecular weight of 74 000 ± 1 400. The amino acid composition is reported. The enzyme has a broad pH optimum between 7.3 and 8.8 and an absolute requirement for either Mn²⁺ or Mg²⁺ for activity; half-maximal activity was observed at 3.7 μM Mn²⁺ or 89.0 μM Mg²⁺. Michaelis constants for geranyl pyrophosphate and isopentenyl pyrophosphate were 0.44 and 0.94 μM, respectively; the V value for synthesis of farnesyl pyrophosphate from these substrates was 1.1 μmol · min⁻¹ · mg⁻¹. Isopentenyl pyrophosphate inhibited the reaction rates at concentrations above 2 μM when the concentrations of geranyl pyrophosphate were less than 2 μM. The highest concentration of geranyl pyrophosphate tested, 16 μM, showed no inhibition of reaction rates even when the concentration of isopentenyl pyrophosphate was as low as 0.2 μM. Only one form of human liver prenyltransferase could be observed under conditions which resolved the porcine enzyme into two distinct forms; the human enzyme is akin, physico-chemically, to the B-form of the pig liver enzyme. After dialysis against Tris-HCl buffer, pH 7.8, the enzyme became completely dependent upon dithiols or thiols for its activity. Kinetic experiments with a partially activated enzyme sample showed that the activation by the dithiol greatly enhanced the affinity of the enzyme for geranyl pyrophosphate, but not that for isopentenyl pyrophosphate. The human prenyltransferase is inactivated by phenylglyoxal according to pseudo-first-order kinetics, but is protected against the inactivation by 3,3-dimethylallyl and geranyl pyrophosphate. It is also inactivated by high concentrations (>2 mM) of iodoacetic acid, but is protected against the inactivation by dithiothreitol. Antibodies raised to the B-form of the pig liver enzyme cross-reacted with the human prenyltransferase and were 47% as effective in precipitating the human enzyme as the porcine enzyme. In double immunodiffusion experiments the antiserum was monospecific against the B-form of the porcine enzyme; it also gave a single precipitin line with the A-form, but not identical with that given by the B-form. It gave a precipitin line also with the human enzyme, but not identical with that given by either the A- or B-form of the porcine enzyme.