J. Basran et al., EFFECTS OF SINGLE-RESIDUE SUBSTITUTIONS ON NEGATIVE COOPERATIVITY IN LIGAND-BINDING TO DIHYDROFOLATE-REDUCTASE, Protein engineering, 10(7), 1997, pp. 815-826
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.