Channeling efficiency in the bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase domain: the effects of site-directed mutagenesis of NADP binding residues.

The three-dimensional structure of the dehydrogenase-cyclohydrolase bifunctional domain of the human trifunctional enzyme indicates that Arg-173 and Ser-197 are within 3 A of the 2'-phosphate of bound NADP. Site-directed mutagenesis confirms that Arg-173 is essential for efficient binding and cannot be substituted by lysine. R173A and R173K have detectable dehydrogenase activity, but the K(m) values for NADP are increased by at least 500-fold. The S197A mutant has a K(m) for NADP that is only 20-fold higher than wild-type, indicating that it plays a supporting role. Forward and reverse cyclohydrolase activities of all the mutants were unchanged, except that the reverse cyclohydrolase activity of mutants that bind NADP poorly, or lack Ser-197, cannot be stimulated by 2',5'-ADP. The 50% channeling efficiency in the forward direction is not improved by the addition of exogenous NADPH and cannot be explained by premature dissociation of the dinucleotide from the ternary complex. As well, channeling is unaffected in mutants that exhibit a wide range of dinucleotide binding. Given that dinucleotide binding is unrelated to substrate channeling efficiency in the D/C domain, we propose that the difference in forward and reverse channeling efficiencies can be explained solely by the movement of the methenylH(4)folate between two overlapping subsites to which it has different binding affinities.