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Abstract

Four different dehydrogenases are known that catalyse the reversible dehydrogenation of N⁵,N¹⁰‐ethylenetetrahydromethanopterin (methylene‐H₄MPT) or N⁵,N¹⁰‐methylenetetrahydrofolate (methylene‐H₄F) to the respective N⁵,N¹⁰‐methenyl compounds. Sequence comparison indicates that the four enzymes are phylogenetically unrelated. They all catalyse the Re‐face‐stereospecific removal of the pro‐R hydrogen atom of the coenzyme's methylene group. The Re‐face stereospecificity is in contrast to the finding that in solution the pro‐S hydrogen atom of methylene‐H₄MPT and of methylene‐H₄F is more reactive to heterolytic cleavage. For a better understanding we determined the conformations of methylene‐H₄MPT in solution and when enzyme‐bound by using NMR spectroscopy and semiempirical quantum mechanical calculations. For the conformation free in solution we find an envelope conformation for the imidazolidine ring, with the flap at N¹⁰. The methylene pro‐S C−H bond is anticlinal and the methylene pro‐R C−H bond is synclinal to the lone electron pair of N¹⁰. Semiempirical quantum mechanical calculations of heats of formation of methylene‐H₄MPT and methylene‐H₄F indicate that changing this conformation into an activated one in which the pro‐S C−H bond is antiperiplanar, resulting in the preformation of the leaving hydride, would require a ΔΔequation image of +53 kJ mol⁻¹ for methylene‐H₄MPT and of +51 kJ mol⁻¹ for methylene‐H₄F. This is almost twice the energy required to force the imidazolidine ring in the enzyme‐bound conformation of methylene‐H₄MPT (+29 kJ mol⁻¹) or of methylene‐H₄F (+35 kJ mol⁻¹) into an activated conformation in which the pro‐R hydrogen atom is antiperiplanar to the lone electron pair of N¹⁰. The much lower energy for pro‐R hydrogen activation thus probably predetermines the Re‐face stereospecificity of the four dehydrogenases. Results are also presented explaining why the chemical reduction of methenyl‐H₄MPT⁺ and methenyl‐H₄F⁺ with NaBD₄ proceeds Si‐face‐specific, in contrast to the enzyme‐catalysed reaction.