Role of S65, Q67, I68, and Y69 Residues in Homotetrameric R67 Dihydrofolate Reductase

Abstract

R67 dihydrofolate reductase (DHFR) shares no sequence or structural homology with chromosomal DHFRs. This enzyme arose recently in response to the clinical use of the antibacterial drug trimethoprim. R67 DHFR is a homotetramer possessing a single active site pore. A high-resolution crystal structure shows the homotetramer possesses exact 222 symmetry [Narayana, N., et al. (1995) Nat. Struct. Biol. 2, 1018-1025]. This symmetry dictates four symmetry-related binding sites must exist for each substrate as well as each cofactor. Isothermal titration calorimetry studies, however, indicate only two molecules bind: either two dihydrofolate molecules, two NADPH molecules, or one substrate and one cofactor [Bradrick, T. D., et al. (1996) Biochemistry 35, 11414-11424]. The latter is the productive ternary complex. To evaluate the role of S65, Q67, I68, and Y69 residues, located near the center of the active site pore, site-directed mutagenesis was performed. One mutation in the gene creates four mutations per active site pore which typically result in large cumulative effects. Steady state kinetic data indicate the mutants have altered K(m) values for both cofactor and substrate. For example, the Y69F R67 DHFR displays an 8-fold increase in the K(m) for dihydrofolate and a 20-fold increase in the K(m) for NADPH. Residues involved in ligand binding in R67 DHFR display very little, if any, specificity, consistent with their possessing dual roles in binding. These results support a model where R67 DHFR utilizes an unusual "hot spot" binding surface capable of binding both ligands and indicate this enzyme has adopted a novel yet simple approach to catalysis.

“Catch 222,” the Effects of Symmetry on Ligand Binding and Catalysis in R67 Dihydrofolate Reductase as Determined by Mutations at Tyr-69

“…It is therefore tempting to suggest that this topology may be preferred in the transition state. These data continue to support the model that Y69F mutations in the homotetramer destabilize binding in the ground state to a greater extent than binding in the transition state (9). Further, the model can be extended such that the Y69F:1ϩ4 double mutant topology appears to provide the least perturbation to ground state binding coupled with the best match to the transition state configuration.…”

“…Docking studies also predict a possible interaction between the tyrosine hydroxyl and the glutamic acid tail of folate. Kinetic studies of the homotetrameric Y69F mutant also support the hypothesis that Tyr-69 interacts with both DHF and NADPH, because the K m values for DHF and NADPH are 11-and 17-fold weaker, respectively, than those for wild-type R67 DHFR (9). In addition, the k cat for the reaction is 2-fold greater than the k cat for wild-type R67 DHFR.…”

“…Those residues identified as most important in R67 DHFR catalysis include 9,12). The first asymmetric mutation series constructed to probe binding and catalysis involved the Q67H substitution (10).…”

“…Transcription and translation yield a monomeric protein (named "Quad3") mimicking the wild-type enzyme. Quad3 is almost fully active (1.8-fold decrease in k cat /K m values), and all physical, binding, and steady-state kinetic studies indicate excellent agreement with wt R67 DHFR behavior (10, 11).Those residues identified as most important in R67 DHFR catalysis include 9,12). The first asymmetric mutation series constructed to probe binding and catalysis involved the Q67H substitution (10).…”

A Balancing Act between Net Uptake of Water during Dihydrofolate Binding and Net Release of Water upon NADPH Binding in R67 Dihydrofolate Reductase

“…Both ⌬C p values for DHF and NADPH binding are small, negative, and within error of each other. Because a number of the interactions between NADPH and protein or DHF and protein are similar (8,15,78,79), the convergence of the ⌬C p values may describe at some level the ability of symmetry-related sites to accommodate the two different ligands.…”