CRYSTAL-STRUCTURES OF CHICKEN LIVER DIHYDROFOLATE-REDUCTASE - BINARY THIONADP+ AND TERNARY THIONADP+.BIOPTERIN COMPLEXES

The role of the 3'-carboxamide substituent of NADPH in the reduction o f pteridine substrates as catalyzed by dihydrofolate reductase (EC 1.5 .1.3, DHFR) has been investigated by determining crystal structures at 2.3 angstrom of chicken liver DHFR in a binary complex with oxidized thionicotinamide adenine dinucleotide (thioNADP+) and in a ternary com plex with thioNADP+ and biopterin. These structures are isomorphous wi th those previously reported for chicken liver DHFR [Volz, K. W., Matt hews, D. A., Alden, R. A., Freer, S. T ., Hansch, C., Kaufman, B. T., & Kraut, J. (1982) J. Biol. Chem. 257, 2528-2536]. ThioNADPH, which ha s a 3'-carbothioamide substituent in place of a 3'-carboxamide, functi ons very poorly as a coenzyme for DHFR [Williams, T. J., Lee, T. K., & Dunlap, R. B. (1977) Arch. Biochem. Biophys. 181, 569-579; Stone, S. R., Mark, A., & Morrison, J. F. (1984) Biochemistry 23, 4340-4346]. Co mparisons show that, while NADP+ and NADPH bind to DHFR with the pyrid ine ring and 3'-carboxamide coplanar, the thioamide group is twisted b y 23-degrees from the pyridine plane in both the binary and ternary co mplexes. This twist appears to be due to steric conflict between the t hioamide sulfur atom and both the pyridine ring at C4 and the adjacent protein backbone at Ala-9. It results in an unfavorably close contact between the sulfur and the biopterin pteridine ring (0.9 angstrom les s than the van der Waals separation) which, on the basis of there fine d structure, greatly destabilizes the binding of biopterin. We infer t hat the thioamide in this position interferes with the transfer of a h ydride ion from thioNADPH to the pteridine substrate by impeding movem ent of the coenzyme pyridine ring and substrate pteridine ring toward one another in the transition state. Thus, the planar conformation of the nicotinamide of NADPH when bound to DHFR appears to minimize steri c repulsion during substrate binding to the holoenzyme complex and dur ing transition-state formation.