HYDROGEN ISOTOPE EFFECTS IN THE REACTIONS CATALYZED BY H-2-FORMING N-5,N-10-METHYLENETETRAHYDROMETHANOPTERIN DEHYDROGENASE FROM METHANOGENIC ARCHAEA

H-2-forming N-5,N-10-methylenetetrahydromethanopterin dehydrogenase from methanogenic Archaea, which is a novel hydrogenase containing neither nickel nor iron-sulfur clusters, catalyzes the reversible reduction of N-5,N-10-methenyltetrahydomethanopterin (CH drop H(4)MPT(+)) with H-2 to N-5,N-10-methylenetetrahydromethanopterin (CH2=H(4)MPT) and a proton (Delta G(o)' = -5.5 kJ/mol). The enzyme also catalyzes a CH drop H(4)MPT(+)-dependent H-2/H+ exchange. We report here on kinetic deuterium isotope effects in these reactions. When CH drop H(4)MPT(+) reduction was perfomed with D-2 instead of H-2, V-max and the K-m did not change. A primary isotope effect of 1 was found al all pH and temperatures tested and independent of whether H2O or D2O was the solvent. The findings indicate that a step other than the activation of H-2 was rate-determining in CH drop H(4)MPT(+) reduction with H-2. This was substantiated by the observation that also the CH drop H(4)MPT(+)-dependent H-2/H+ exchange reaction did not exhibit an appreciable deuterium isotope effect. V-max for CH2=H(4)MPT dehydrogenation to CH drop H(4)MPT(+) and H-2 was only 2-3 times higher than for CD2=H(4)MPT dehydrogenation to CD drop H(4)MPT(+) and HD. Such a small primary isotope effect indicates that the breakage of the C-H bond in the methylene group of CH2=H(4)MPT was only rate-limiting when hydrogen was substituted by a deuterium.