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Site‐specific mutagenesis of dihydrofolate reductase from Escherichia coli
Author(s) -
Chen JinTann,
Mayer Ruth J.,
Fierke Carol A.,
Benkovic Stephen J.
Publication year - 1985
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.240290203
Subject(s) - dihydrofolate reductase , escherichia coli , chemistry , dissociation constant , stereochemistry , enzyme , mutant , reaction rate constant , cofactor , active site , dissociation (chemistry) , pyrophosphate , biochemistry , kinetics , organic chemistry , gene , physics , receptor , quantum mechanics
Two site‐specific mutations of dihydrofolate reductase from Escherichia coli based on the x‐ray crystallographic structure were constructed. The first mutation (His‐45 → Gln) is aimed at assessing the interaction between the imidazole moiety and the pyrophosphate backbone of NADPH. The second (Thr‐113 → Val) is part of a hydrogen bonding network that contacts the dihydrofolate substrate and may be involved in proton delivery to the N5‐;C6 imine undergoing reduction. The first mutation was shown to alter both the association and dissociation rate constants for the cofactor so that the dissociation constant was increased 6–40‐fold. A corresponding but smaller (fourfold) effect was noted in V/K but not in V compared to the wild‐type enzyme. The second was demonstrated to increase the dissociation rate constant for methotrexate 20–30‐fold, and presumably dihydrofolate also, with it corresponding 20–30‐fold increase in the dissociation constant. In this case an identical effect was noted on V/K but not in V relative to the native enzyme. Thus, in both mutant enzymes the decrease in binding has not been translated into a loss of catalytic efficiency.