Nuclear magnetic resonance study of the state of protonation of inhibitors bound to mutant dihydrofolate reductase lacking the active-site carboxyl

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Abstract
13C nuclear magnetic resonance spectra have been obtained for complexes of [2-13C]methotrexate and [2-13C]trimethoprim with wild-type dihydrofolate reductase (DHRF) from Escherichia coli and with two mutant enzymes in which aspartic acid-27 is replaced by asparagine and by serine, respectively. In both the wild-type and mutated enzymes, exchange between the free inhibitor and the enzyme-complexed inhibitor is slow on the NMR times scale; hence, despite the considerably increased dissociation constants for binary complexes with the enzymes, the dissociation rate remains small relative to the frequency separation of the resonances. In all cases but one, the pKa of an inhibitor that is complexed to enzyme differs greatly from that of the free inhibitor. However, while the pKa of both inhibitors in complexes with the wild-type enzyme is elevated to above 10, the pKa of the inhibitors complexed with the Asn-27 and Ser-27 enzymes is lowered to a value below 4. Exact determinations of bound pKa values were limited by the solubility of the enzyme and the dissociation constants of the complexes. The single exception to these general conclusions is the ternary complex of the Ser-27 DHFR with trimethoprim and NADPH. In this complex, both free and enzyme-complexes trimethoprim exhibit similar pKa values (.apprxeq. 7.6). However, both the exchange between free and enzyme-complexed inhibitor and the protonation of the enzyme-complexed inhibitor are slow in the NMR time scale, so that the spectra reveal three resonances corresponding to free inhibitor, to protonated enzyme-complexed inhibitor, and to unprotonated enzyme-complexed inhibitor. Evidence for another slow conformational transition of the enzyme was also obtained in studies of the binary complex of Asn-27 DHFR with [2-13C]methotrexate. These results are discussed in terms of crystallographic studies recently carried out on these mutated enzymes [Howell, E. E., Villafranca, J. E., Warren, M. S., Oatley, S. J., and Kraut, J. (1986) Science (Washington, D. C.) 231, 1123-1128].

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