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 bifunct ional domain of the human trifunctional enzyme indicates that Arg-173 and S er-197 are within 3 Angstrom of the 2'-phosphate of bound NADP. Site-direct ed mutagenesis confirms that Arg-173 is essential for efficient binding and cannot be substituted by lysine. R173A and R173K have detectable dehydroge nase activity, but the K-m values for NADP are increased by at least 500-fo ld. The S197A mutant has a K-m for NADP that is only 20-fold higher than wi ld-type, indicating that it plays a supporting role. Forward and reverse cy clohydrolase activities of all the mutants were unchanged, except that the reverse cyclohydrolase activity of mutants that bind NADP poorly, or lack S er-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 a nd cannot be explained by premature dissociation of the dinucleotide from t he ternary complex. As well, channeling is unaffected in mutants that exhib it 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 over lapping subsites to which it has different binding affinities. (C) 2000 Els evier Science B.V. All rights reserved.