Crystal structures of E. coli and DHFR

Yong-Qing Chen

Appointment Period: 1995-1996 / Grant Year: [10]

Explorations of the relationship between macromolecular structure and function are a key element of modern biochemistry. Our group uses a combination of X-ray crystallography and site-directed mutagenesis to examine this relationship. Determination of a target enzyme's structure provides the starting point. This is followed by iterative cycles of site-directed mutagenesis and characterization of the effects of the induced mutation on enzyme structure and function. In this way it is possible to identify residues that are critical for catalysis; to evaluate the energetics of enzyme:substrate interactions; to examine the effect of the local environment on reactivity: and to engineer new or modified reactivities into the parent enzyme.

During my postdoctoral research, I have been investigating the catalytic mechanism of dihydrofolate reductase (DHFR), which is of tremendous importance as a target enzyme for chemotherapeutics. The X-ray crystallographic structures of DHFR from Escherichia coli have been solved as binary complexes with NADP+ and NADPH4 (an analog of NADPH). Many interesting features are observed from these structures. They provide a basis for a new theory concerning the mechanism of dihydrofolate protonation and stabilization, which are long-standing problems in structural studies of DHFR.

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (H-2folate) to tetrahydrofolate by NADPH, and this requires that the pteridine ring be protonated at N5. A long-standing puzzle has been how, at physiological pH, the enzyme can protonate N5 in view of its solution pK-a of 2.6 and the fact that the only proton-donating group in the pteridine binding site, Asp-27, hydrogen bonds not to N5 but to the 2-amino group and N3 of the pterin ring. We have determined the pK-a of N5 of dihydrofolate in the Escherichia coli DHFR/NADP+/H-2folate ternary complex by Raman difference spectroscopy and found that the value is 6.5. In contrast, the pK-a of N5 is less than 4.0 in either the binary complex, the ternary complex with an analogue of NADPH (H-2NADPH), or the Asp27 to serine mutant DHFR (D27S) ternary complex with NADP+. Thus, one need not invoke proton donation from Asp-27 to N5 via a series of bound water molecules and/or pteridine-ring substituents. We propose instead that the N5 protonated form of H-2folate is stabilized directly at the active site in the DHFR/NADPH/H-2folate complex by specific interactions that form only in the ternary complex, involving perhaps a bound water molecule, the carboxamide moiety of the coenzyme, and/or the local electrostatic field of the enzyme molecule, to which an important contribution may be made by Asp-27.

PUBLICATIONS (resulting from this training)

Chen YQ, Kraut J, Blakley RL, Callender R. (1994) Determination by Raman spectroscopy of the pKa of N5 of dihydrofolate bound to dihydrofolate reductase: mechanistic implications. Biochemistry 33:7021-6.

Chen YQ, Kraut J, Callender R. (1997) pH-dependent conformational changes in Escherichia coli dihydrofolate reductase revealed by Raman difference spectroscopy. Biophys J. 72:936-41.