The role of conserved residues in Lactobacillus casei dihydrofolate reductase.

Mutants of three conserved residues in Dihydrofolate reductase (DHFR) from Lactobacillus casei have been studied to assess their roles in ligand binding and catalysis. Aspartate26 has been widely postulated to be the source of the proton in the reaction catalysed by DHFR; this was probed by the Aspartate26/Asparagine (D26N) mutation. The rate of hydride ion transfer, which governs kcat in the D26N mutant, is reduced by a factor of 10 (pH 7.5) when compared to the wild-type enzyme. The results argue against the role of Aspartate26 as the primary proton donor, but may be more consistent with a mechanism whereby it promotes enolisation of the substrate during the reaction. The Tryptophan21/Histidine (W21H) mutant binds the coenzyme NADPH over 1000-fold more weakly than wild-type DHFR. The magnitude of the negative cooperative effect between NADPH and FH4 (a crucial feature of the kinetic scheme of DHFR), has been greatly reduced in the W21H mutant, suggesting an important role for Tryptophan21 in the mechanism of negative cooperativity. The pH dependence of kcat for W21H (which is equivalent to the rate of hydride ion transfer) has a form that reflects the cooperative ionisation of two groups. Kinetic and NMR results suggest that the new Histidine21 is one of the groups responsible for the unusual ionisation curve. Substrate, inhibitor and coenzyme binding are unaffected by the Arginine57/Lysine (R57K) mutation. With the wild-type enzyme, loss of the ion-pair interaction between MTX and Arginine57 also leads to a loss of the interaction with Histidine28; this is not the case with the R57K mutant. The Arginine57/Lysine substitution has little effect on the rate of catalysis although the apparent pKa of kcat is reduced by 0.6 units, despite the site of catalysis being more than 15A away from the site of the mutation. The origin of this effect may be due to electrostatic or structural factors.