19F-n.m.r. studies of 3′,5′-difluoromethotrexate binding to Lactobacillus casei dihydrofolate reductase. Molecular motion and coenzyme-induced conformational changes

1 February 1984

journal article
research article
Published by Portland Press Ltd. in Biochemical Journal

Vol. 217 (3) , 659-666
https://doi.org/10.1042/bj2170659

Abstract

19F-n.m.r. spectroscopy was used to study the binding of 3′,5′-difluoromethotrexate to dihydrofolate reductase (tetrahydrofolate dehydrogenase) from Lactobacillus casei. The benzoyl ring of the bound difluoromethotrexate was found to ‘flip’ about its symmetry axis, and the rate (7.3 × 10(3) s-1 at 298 K) and activation parameters for this process were determined by lineshape analysis of the 19F-n.m.r. spectrum at a series of temperatures in the range 273-308 K. The contributions to the barrier for this process are discussed. Addition of NADP+ or NADPH to form the enzyme-difluoromethotrexate-coenzyme ternary complex led to an increase in the rate of benzoyl ring flipping by a factor of 2.6-2.8-fold, and to substantial changes in the 19F-n.m.r. chemical shifts. The possible nature of the coenzyme-induced conformational changes responsible for these effects is discussed.

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