Role of Lys-32 Residues in R67 Dihydrofolate Reductase Probed by Asymmetric Mutations

Abstract

R67 dihydrofolate reductase (R67 DHFR) is a novel protein encoded by an R-plasmid that confers resistance to the antibiotic, trimethoprim. This homotetrameric enzyme possesses 222 symmetry, which imposes numerous constraints on the single active site pore, including a "one-site-fits-both" strategy for binding its ligands, dihydrofolate (DHF) and NADPH. Previous studies uncovered salt effects on binding and catalysis (Hicks, S. N., Smiley, R. D., Hamilton, J. B., and Howell, E. E. (2003) Biochemistry 42, 10569 -10578), however the one or more residues that participate in ionic contacts with the negatively charged tail of DHF as well as the phosphate groups in NADPH were not identified. Several studies predict that Lys-32 residues were involved, however mutations at this residue destabilize the R67 DHFR homotetramer. To study the role of Lys-32 in binding and catalysis, asymmetric K32M mutations have been utilized. To create asymmetry, individual mutations were added to a tandem array of four in-frame gene copies. These studies show one K32M mutation is tolerated quite well, whereas addition of two mutations has variable effects. Two double mutants, K32M:1؉2 and K32M: 1؉4, which place the mutations on opposite sides of the pore, reduce k cat . However a third double mutant, K32M: 1؉3, that places two mutations on the same half pore, enhances k cat 4-to 5-fold compared with the parent enzyme, albeit at the expense of weaker binding of ligands. Because the k cat /K m values for this double mutant series are similar, these mutations appear to have uncovered some degree of non-productive binding. This non-productive binding mode likely arises from formation of an ionic interaction that must be broken to allow access to the transition state. The K32M:1؉3 mutant data suggest this interaction is an ionic interaction between Lys-32 and the charged tail of dihydrofolate. This unusual catalytic scenario arises from the 222 symmetry imposed on the single active site pore. R67 dihydrofolate reductase (R67 DHFR)1 is an R-plasmidencoded enzyme that catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate. Its presence in bacteria confers resistance to the antibiotic, trimethoprim. This enzyme is not similar in sequence or structure to the chromosomally encoded DHFRs.R67 DHFR is a homotetramer, and the pore that traverses the length of the molecule is the active site. Surprisingly, the structure possesses 222 symmetry (1), which imposes numerous constraints on binding and catalysis. For example, the symmetry requires that for each binding site, there must be three additional, symmetry-related sites. However, solution studies find only two sites can be occupied simultaneously because of steric constraints. The possible binding combinations are two NADPH molecules, or two folate/DHF molecules, or one NADPH plus one folate/DHF molecule (2). Only the latter is productive. Thus binding of neither ligand can be optimized, and a "one-site-fits-both" approach is employed (3, 4). Another constraint arising from ...

Searching Sequence Space: Two Different Approaches to Dihydrofolate Reductase Catalysis

“…Loss of an ionic interaction between a symmetry-related K32 residue in the second half of the pore with the Glu tail of DHF might be the key event that facilitates catalysis. [42] This scenario could permit the ligands to move towards a position associated with the correct distance and angle for hydride transfer as well as to exclude solvent. This unusual mechanism likely arises from the need to balance catalysis with the constraints imposed by the 222 symmetry of the active site pore.…”

“…Breaking the symmetry of R67 DHFR by introduction of asymmetric mutations suggests one role of the symmetry is to provide an avidity or multivalency effect. [40,42,43] Here, once a site is occupied, the proximity of other symmetry-related sites can enhance binding by reduction of the associated entropy and/or by decreasing the dissociation rate. [44][45][46] This section illustrates that active-site mutants of Ec DHFR mostly have local effects on ligand binding.…”

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

“…Previous crystallography and NMR studies (8,9,11,12) show NADPH binds in an extended conformation, with numerous specific interactions, including H-bonds between the nicotinamide carboxamide of NADP ϩ with backbone NH and O atoms from Ile-68. Ionic interactions also form between the nicotinamide phosphate and the adenosyl-2Ј-phosphate with symmetry-related Lys-32 residues (13)(14)(15)(16).…”

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

“…The asymmetric Y69F mutants were not as prone to aggregation as the asymmetric K32M mutants (companion paper, Hicks et al (42)); however, visible turbidity was sometimes noted at the high protein concentrations (ϳ100 M) used in isothermal titration calorimetry experiments. Thus 0.1 g/liter polyethylene glycol 3350 was added to minimize aggregation as well as increase purification yields of the asymmetric mutants (16).…”