EFFECTS OF SINGLE-RESIDUE SUBSTITUTIONS ON NEGATIVE COOPERATIVITY IN LIGAND-BINDING TO DIHYDROFOLATE-REDUCTASE

The effects of six amino acid substitutions in Lactobacillus casei dih ydrofolate reductase, predominantly in the coenzyme binding site, on c atalysis and on the negative cooperativity between NADPH and tetrahydr ofolate binding have been determined. Replacement of Leu62, His64 or L eu54 by alanine has no effect on k(cat), and produces only modest chan ges in negative cooperativity. Alanine substitution of His77, which in teracts indirectly with the coenzyme adenine ring, leads to a doubling of the negative cooperativity and a consequent doubling of k(cat). Re placement of Arg43, which interacts with the coenzyme 2'-phosphate, by alanine, or of Trp21, which interacts with the coenzyme nicotinamide ring, by histidine leads to a 20-100-foId decrease in negative coopera tivity. In both mutants there is a decrease in k(cat); isotope effects show that product release is largely rate-limiting in R43A, whereas i n W21H hydride ion transfer is rate-limiting, H-1 NMR has been used to obtain information on the extent of the structural changes produced b y the substitutions. This varies from very local effects in H64A to ve ry widespread effects in W21H, These changes are used as the basis for discussion of the mechanisms of the functional effects of the various substitutions, It is suggested that residues in helix C, beta-strand 3 and the beta 3-beta 4 loop may be involved in the transmission of ef fects between the coenzyme and substrate binding sites.