Crystal structures of chicken liver dihydrofolate reductase: Binary thioNADP+ and ternary thioNADP+.cntdot.biopterin complexes

Abstract

The role of the 3'-carboxamide substituent of NADPH in the reduction of pteridine substrates as catalyzed by dihydrofolate reductase (EC 1.5.1.3, DHFR) has been investigated by determining crystal structures at 2.3 A of chicken liver DHFR in a binary complex with oxidized thionicotinamide adenine dinucleotide (thioNADP+) and in a ternary complex with thioNADP+ and biopterin. These structures are isomorphous with those previously reported for chicken liver DHFR [Volz, K.W., Matthews, D.A., Alden, R.A., Freer, S. T., Hansch, C., Kaufman, B. T., & Kraut, J. (1982) J. Biol. Chem. 257, 2528-2536]. ThioNADPH, which has a 3'-carbothioamide substituent in place of a 3'-carboxamide, functions 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]. Comparisons show that, while NADP+ and NADPH bind to DHFR with the pyridine ring and 3'-carboxamide coplanar, the thioamide group is twisted by 23 degrees from the pyridine plane in both the binary and ternary complexes. This twist appears to be due to steric conflict between the thioamide 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 A less than the van der Waals separation) which, on the basis of the refined structure, greatly destabilizes the binding of biopterin.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in a Conformational Equilibrium Distinguish Catalysis by the Endothelial and Neuronal Nitric-oxide Synthase Flavoproteins

“…This single atom substitution relative to NADP ϩ can result in altered binding characteristics in nucleotide-binding proteins (51,52,66), and it may also cause larger absorbance shifts near 510 nm (50, 67). 4 We encountered some difficulty with excessive equilibration/stabilization times for the FMN ox/sq couple during oxidative titrations of eNOSr.…”

Interactions between Transhydrogenase and Thio-nicotinamide Analogues of NAD(H) and NADP(H) Underline the Importance of Nucleotide Conformational Changes in Coupling to Proton Translocation

“…However, when these two proteins are loaded with thio-NADP ϩ , the carbothiamide group of the analogue in both cases is twisted relative to the plane of the pyridine ring, although the twist has the opposite sense: X am is Ϫ160°in DHFR and ϩ140°in dIII. The increased twist of the carbothioamide group in DHFR was suggested to avoid unfavorable contact between the sulfur atom and the CH group at position 4 of the pyridine ring (20). It was noted to be less than in a model compound, 2-methyl-4(thiocarbamoyl)-pyridine (47).…”

“…It was noted to be less than in a model compound, 2-methyl-4(thiocarbamoyl)-pyridine (47). McTigue et al (20) concluded that a larger rotation in DHFR is prohibited by the binding site geometry of the protein and that the unfavorable interactions between the CH group of pyridine ring and the S atom are not completely relieved. The oppositely directed twist of the carbothiamide group in dIII (Fig.…”

“…7). In the crystal structure of a ternary complex of DHFR, the rotated sulfur atom in the thio-NADP ϩ results in destabilization of the bound biopterin; the latter is moved away from the nicotinamide ring, and its temperature factors are increased relative to those in the NADP ϩ structure (20). This suggested an explanation for the finding that thio-NADPH is a very poor coenzyme for DHFR; in the formation of the transition state for hydride transfer, the mutual approach of the nicotinamide ring and the substrate pteridine ring are impeded by the sulfur atom of the thioamide.…”

“…The binding properties of the analogues can be different relative to those of the physiological nucleotides and the catalytic rate can be affected; both increases and decreases have been observed in different enzymes (19). An x-ray structure of dihydrofolate reductase (DHFR) 1 with bound thio-NADP ϩ showed how small distortions of the nucleotide conformation can lead to pronounced effects on catalysis (20). Because the absorbance band of its reduced form is redshifted relative to that of the physiological substrate (and therefore has minimal spectral overlap with NADH), thio-NADP ϩ has often been used to monitor the activity of proton-translocating transhydrogenase (21), but few experiments have been carried out using thio-nicotinamide analogues with a view to elucidating mechanistic details of the enzyme.…”