Nuclear magnetic resonance study of dihydrofolate reductase labeled with [.gamma.-13C]tryptophan

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Abstract
Dihydrofolate reductase (EC 1.5.1.3) isozyme 2 of Streptococcus faecium was labeled with 13C in the C.gamma. position of tryptophan residues by growing the organism on a defined medium containing L-[.gamma.-13C]tryptophan (90% 13C). The 13C NMR spectrum of the enzyme shows 4 well-resolved resonances which have nuclear Overhauser enhancements of 1.1-1.3. Values of T1 (spin-lattice relaxation time) and T2 (spin-spin relaxation time) are significantly less than predicted for an isotropically rotating, rigid sphere with no intermolecular dipole-dipole interactions. Three of the resonances have chemical shifts downfield from the 13C resonance of urea-denatured enzyme by amounts up to 1.43 ppm. The chemical shift of resonance 4 in the spectrum is 4.0 ppm upfield from Trp C.gamma. of urea-denatured enzyme. This large upfield shift is attributed to electric field effects generated by polar side chains. The 2 more upfield peaks both provide evidence that the corresponding tryptophan residues, WC and WD, each undergo chemical exchange between alternative microenvironments. In the case of WC, which gives a resonance with 2 components, exchange is slow (ve, exchange rate < 55 s-1), and the relative populations of the 2 stable states are in the ratio 2:3. WD is apparently in intermediate to fast exchange on the NMR time scale. With a 2-state model, ve increases from 90 to 150 s-1 as the temperature is increased from 5 to 25.degree. C. This increase in temperature is also accompanied by an increase in the fractional population of the minor downfield state(s), from about 0.062 at 5.degree. C to 0.24 at 25.degree. C. The data may also be interpreted as a temperature-dependent equilibrium between a continuum of many states. WD is tentatively identified with Trp-22 since comparison of the sequences of Lactobacillus casei dihydrofolate reductase and S. faecium dihydrofolate reductase and inspection of the crystal structure of the L. casei enzyme indicate that Trp-6, Trp-115 and Trp-160 are probably all involved in regions of .beta.-sheet whereas Trp-22 is in a loop joining .beta.A to .alpha.B. Earlier crystallographic evidence for the Escherichia coli reductase suggests that in the methotrexate complex with this enzyme the corresponding loop has a good deal of flexibility. In the uncomplexed S. faecium reductase the motion of this loop is probably the major mechanism for the exchange process involving Trp-22. The upfield chemical shift of resonance 4 is attributed to electric field effects on C.gamma. of Trp-22 produced by the carboxylate groups of Asp-27 and Asp-9. On the basis of the small difference between the chemical shift of resonance 3 and that of tryptophan C.gamma. in urea-denatured reductase, it is suggested that WC may be identified with Trp-6.

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