Re-face stereospecificity of methylenetetrahydromethanopterin and methylenetetrahydrofolate dehydrogenases is predetermined by intrinsic properties of the substrate

Four different dehydrogenases are known that catalyse the reversible dehydr ogenation of N-5,N-10-methylenetetrahydrometha-nopterin (methylene-H4MPT) o r N-5,N-10-methylenetetrahydrofolate (methylene-H4F) to the respective (NN1 0)-N-5-methenyl compounds. Sequence comparison indicates that the four enzy mes are phylogenetically unrelated They all catalyse the Re-face-stereospec ific 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 solutio n the pro-S hydrogen atom of methytene-H4MPT and of methylene-H4F is more r eactive 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. F or the conformation free in solution we find an envelope conformation for t he imidazoline ring, with the flop at N-10. The methylene pro-S C-H bond is anticlinal and the methylene pro-R C-H bond is synclinal to the lone elect ron pair of N-10. 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 antiperi planar, resulting in the preformation of the leaving hydride, would require a Delta Delta (f)degrees of +53 kJ mol(-1) for methylene-H4MPT and of +57 kJ mol(-1) for methylene-H4F This is almost twice the energy required to fo rce the imidazolidine ring in the enzyme-bound conformation of methylene-H4 MPT (+29 kJ mol(-1)) or of methylene-HF (+35 kJ mol(-1)) into an activated conformation in which the pro-R hydrogen atom is antiperiplanar to the lone electron pair of N-10. The-much lower energy for pro-R hydrogen activation thus probably predetermines the Re-face stereospecificity of the four dehy drogenoses. Results are also presented explaining why the chemical reductio n of methenyl-H4MPT+ and methenyl-H4F+ with NaBD4 proceeds Si-face-specific , in contrast to the enzyme-catalysed reaction.