Functional Role of a Mobile Loop of Escherichia coli Dihydrofolate Reductase in Transition-State Stabilization

Luyuan Li, Christopher J. Falzone, Stephen J. Benkovic, Peter E. Wright

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Abstract

The function of a highly mobile loop in Escherichia coli dihydrofolate reductase was studied by constructing a mutant (DL1) using cassette mutagenesis that had four residues deleted in the middle section of the loop (Metl6-Alal9) and a glycine inserted to seal the gap. This part of the loop involves residues 16-20 and is disordered in the X-ray crystal structures of the apoprotein and the NADP+binary complex but forms a hairpin turn that folds over the nicotinamide moiety of NADP+and the pteridine moiety of folate in the ternary complex [Bystroff, C., & Kraut, J. (1991) Biochemistry 30, 2227-2239]. The steady-state and pre-steady-state kinetics and two-dimensional ‘H NMR spectra were analyzed and compared to the wild-type protein. The kinetics on the DL1 mutant enzyme show that the KM value for NADPH (5.3 µM), the KM for dihydrofolate (2 µM), the rate constant for the release of the product tetrahydrofolate (10.3 s-1). and the intrinsic pKa value (6.2) are similar to those exhibited by the wild-type enzyme. However, the hydride-transfer rate declines markedly from the wild-type value of 950 s-1to 1.7 s-1for the DL1 mutant and when taken with data for substrate binding indicates that the loop contributes to substrate flux by a factor of 3.5 X 104. Thus, the mobility of loop I may provide a mechanism of recruiting hydrophobic residues which can properly align the nicotinamide and pteridine rings for the hydride-transfer process (a form of transition-state stabilization). Two-dimensional NMR spectra show a slow (on the NMR time scale) exchange process at 298 K in the wild-type protein which cannot be detected in the deletion mutant. This slow process in the wild-type protein can be partially attributed to a two-site exchange of some of the residues in loop I. Therefore, the deletion of the hairpin-forming residues in loop I also affects the observed dynamics of the secondary structure in the wild-type protein and thus ties the changes in the kinetic behavior of the DL1 protein to the restricted movement of its loop I relative to the wild-type enzyme.

Original languageEnglish (US)
Pages (from-to)7826-7833
Number of pages8
JournalBiochemistry
Volume31
Issue number34
DOIs
StatePublished - Feb 1 1992

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All Science Journal Classification (ASJC) codes

  • Biochemistry

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