Binding and interconversion of tetrahydrofolates at a single site in the bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase

Abstract

The bifunctional dehydrogenase/cyclohydrolase domain of the human NADP-dependent trifunctional methyleneH4folate dehydrogenase/methenylH4folate cyclohydrolase/formylH4folate synthetase (H4folate = tetrahydrofolate) catalyzes two sequential reactions involved in the interconversion of H4folate derivatives. We have established by equilibrium dialysis that a single H4folate-binding site exists per monomer of the dimeric domain and that the presence of nucleotides has two unexpected effects on H4folate substrate binding. Nucleotides containing a 5'-phosphate cause positive cooperativity in the binding of methyleneH4folate but not of 10-formylH4folate, and NADP increases the affinity for 10-formylH4folate by a factor of 25. The results indicate that dinucleotide preferentially binds before 10-formylH4folate in the reverse cyclohydrolase reaction, and this mechanism increases the efficiency of conversion of 10-formylH4folate to methyleneH4folate. We report new kinetic data that are also consistent with a steady-state random mechanism for this enzyme. To assess whether the enzyme functions at equilibrium in vivo, we determined the overall chemical equilibrium constant of Keq = 16 for ([10- formylH4folate][NADPH])/([methyleneH4folate][NADP]). Using this value and reported ratios of free dinucleotides and folate derivatives in vivo, we estimate that the cytosolic dehydrogenase/cyclohydrolase reactions exist near the equilibrium position. However, the NAD-dependent dehydrogenase/cyclohydrolase reactions in mitochondria are far from equilibrium and are poised toward 10-formylH4folate synthesis. The results of the binding and kinetic studies indicate that the bifunctional nature of the methyleneH4folate dehydrogenase/methenylH4folate cyclohdrolase domain is designed to optimize the overall reverse reactions in vivo.

Mitochondrial MTHFD2L Is a Dual Redox Cofactor-specific Methylenetetrahydrofolate Dehydrogenase/Methenyltetrahydrofolate Cyclohydrolase Expressed in Both Adult and Embryonic Tissues

“…Thus, the ratio of CH 2 -THF to 10-CHO-THF in the matrix (reactions 3m and 2m in Fig. 1) will be shifted much further toward 10-CHO-THF with a CH 2 -THF dehydrogenase linked to the NAD ϩ /NADH pool versus the NADP ϩ /NADPH pool (6,7). A cyclohydrolase/ dehydrogenase with dual cofactor specificity, such as MTHFD2L, would be able to adapt immediately to changing metabolic conditions, shifting the equilibrium between CH 2 -THF and 10-CHO-THF (and formate) depending on the relative levels of oxidized cofactor (NAD ϩ or NADP ϩ ) in the mitochondrial matrix.…”

“…1, reaction 2m). This enzyme, now referred to as MTHFD2, has been extensively characterized with respect to kinetics, substrate specificity, and expression profile (3,(5)(6)(7)(8)(9)(10).…”

The crystal structure of a bacterial, bifunctional 5, 10 methylene‐tetrahydrofolate dehydrogenase/cyclohydrolase

“…Kinetic studies suggest that interaction of the poly-g-glutamyl tail of the methylene-tetrahydrofolate molecule with the enzyme provides binding energy and affinity for the substrate, while allowing dynamic motion and kinetic channeling of the reactive methenylpterin ring system between dehydrogenase and cyclohydrolase active sites~Cohen & MacKenzie, 1978;Wasserman et al, 1983;Green et al, 1988;Hum & MacKenzie, 1991;Pelletier & MacKenzie, 1995!. Inspection of the surface of the E. coli dph0cyc revealed that there are two significant regions of positive-charged residues, one at each end of the cleft between the two a0b domains of deh0cyc.…”

“…The methenyl-THF product of the dehydrogenase reaction displays significant kinetic "channeling" to the cyclohydrolase active site, where it is converted to formyl- THF~Cohen & MacKenzie, 1978;Wasserman et al, 1983;Hum & MacKenzie, 1991;Pelletier & MacKenzie, 1995!. Several chemical studies indicate that the deh and cyc active sites are in close proximity and might overlap~Schirch, 1978; Drummond et al, 1983;Appling & Rabinowitz, 1985!.…”

Reversal of Cytosolic One-Carbon Flux Compensates for Loss of the Mitochondrial Folate Pathway

“…The NADPH/NADP + ratio in turn may play an important role in regulating the cytosolic flux of 1C units through the MTHFD1 dehydrogenase reaction. The relatively high NADPH/NADP + ratio in the cytosol may favor reduction of 1C units from 10-formyl-THF into methylene-THF, whereas low NADH levels in the mitochondria favor the opposite (Pelletier and MacKenzie, 1995).…”