Dihydrofolate reductase hysteresis and its effect on inhibitor binding analyses

Abstract

Escherichia coli dihydrofolate reductase was shown to follow slow transient kinetics (hysteresis). Nonlinear reaction velocities were detected during the enzyme assay and required 10-15 min to reach a steady-state rate. The degree of hysteresis was influenced by the enzyme concentration and the order of substrate addition. Incubation of the enzyme with NADPH before addition of dihydrofolate resulted in slow initial velocities that increased up to 2-fold during the course of the assay. Increasing the enzyme concentration from 0.2 to 1 nM resulted in diminished hysteresis. NADPH-initiated reactions were linear at all enzyme concentrations tested. Certain drugs had profound effects on hysteresis. Pyrimethamine practically eliminated the hysteresis of dihydrofolate-started reactions, whereas trimethoprime augmented the non-linearities in the sense that hysteresis was detected in both enzyme- and NADPH-started reactions. The shape of these reaction tracings makes trimethoprim is not a slow-binding inhibitor when assayed under conditions that eliminate hysteresis. Contrary to this, sulfamethoxazole did not affect hysteresis or augment inhibition of the enzyme by trimethoprim. Sulfamethoxazole alone (at 6 mM) did not inhibit the hysteresis and allow reliable determinations of Ki values of both weak and tight binding inhibitors. For example, Ki values for pyrimethamine, trimethoprim, and methotrexate were found to be 214 nM, 1.3 nM, and 0.021 nM, respectively.

Characterization of the gene for the chromosomal dihydrofolate reductase (DHFR) of Staphylococcus epidermidis ATCC 14990: the origin of the trimethoprim-resistant S1 DHFR from Staphylococcus aureus?

“…The DHFR assays have been described previously (4). The concentration of dihydrofolate was measured spectrophotometrically at 282 nm by using a molar extinction coefficient of 2.8 ϫ 10 4 M Ϫ1 cm Ϫ1 at pH 7.4 (2).…”

Cloning and characterization of a novel, plasmid-encoded trimethoprim-resistant dihydrofolate reductase from Staphylococcus haemolyticus MUR313

“…The DHFR assay has been described previously (4). Dihydrofolate (DHF) was prepared from folic acid, and its concentration was measured spectrophotometrically at 282 nm by using the molar extinction coefficient of 2.8 ϫ 10 4 M Ϫ1 cm Ϫ1 at pH 7.4 (1).…”

Characterization of the gene for chromosomal trimethoprim-sensitive dihydrofolate reductase of Staphylococcus aureus ATCC 25923

“…The DHFR assay has been described previously (1). A unit of DHFR activity was taken as the conversion of 1 ,M dihydrofolate per min.…”