ISSN:
0730-2312
Keywords:
steady state
;
site-specific mutagenesis
;
kinetics
;
dihydrofolate reductase
;
binding
;
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
Notes:
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.
Additional Material:
5 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/jcb.240290203
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