Hydrogen isotope effects in the reactions catalyzed by H2-forming N5,N10-methylenetetrahydromethanopterin dehydrogenase from methanogenic Archaea.
H2-forming N5,N10-methylenetetrahydromethanopterin dehydrogenase from methanogenic Archaea, which is a novel hydrogenase containing neither nickel nor iron-sulfur clusters, catalyzes the reversible reduction of N5,N10-methenyltetrahydomethanopterin (CH identical to H4MPT+) with H2 to N5,N10-methylenetetrahydromethanopterin (CH2 = H4MPT) and a proton (delta G degree' = -5.5 kJ/mol). The enzyme also catalyzes a CH identical to H4MPT(+)-dependent H2/H+ exchange. We report here on kinetic deuterium isotope effects in these reactions. When CH identical to H4MPT+ reduction was performed with D2 instead of H2, Vmax and the Km did not change. A primary isotope effect of 1 was found at 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 H2 was rate-determining in CH identical to H4MPT+ reduction with H2. This was substantiated by the observation that also the CH identical to H4MPT(+)-dependent H2/H+ exchange reaction did not exhibit an appreciable deuterium isotope effect. Vmax for CH2 = H4MPT dehydrogenation to CH identical to H4MPT+ and H2 was only 2-3 times higher than for CD2 = H4MPT dehydrogenation to CD identical to H4MPT+ and HD. Such a small primary isotope effect indicates that the breakage of the C-H bond in the methylene group of CH2 = H4MPT was only rate-limiting when hydrogen was substituted by a deuterium.
Klein
A R
AR
Max-Planck-Institut für terrestrische Mikrobiologie, Philipps-Universität Marburg, Germany.
Hartmann
G C
GC
Thauer
R K
RK
D003903:Deuterium; D019605:Euryarchaeota / Q000201:enzymology*; D006859:Hydrogen; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D017014:Methanobacterium / Q000201:enzymology; D017017:Methanococcus / Q000201:enzymology; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism*; D011522:Protons; D011622:Pterins / Q000737:chemistry / Q000378:metabolism
C091917:N(5),N(10)-methylene-tetrahydromethanopterin; D011522:Protons; D011622:Pterins; D006859:Hydrogen; D003903:Deuterium; D000587:Oxidoreductases Acting on CH-NH Group Donors; C047126:methylenetetrahydromethanopterin dehydrogenase