The Effect of Codon 31 on the Relative Affinities for the Binding of Designed 8-Alkyl-Pterins to Dihydrofolate Reductase: A Statistical Perturbation Theory and Molecular Dynamics Simulation Study
Although a high degree of primary sequence and structural homology exists between rhDHFR (recombinant human dihydrofolate reductase) and clDHFR (chicken liver DHFR), particularly in the active-site region,1,2 kinetic studies of the designed 8-alkyl-pterin substrates appear to have revealed small but consistent differences in the Km’s for these two enzymes,3 with those for clDHFR being the lower. In contrast, the dissociation constants (Kd’s) for the binding of a number of 8-alkyl-N5-deazapterin inhibitors indicate a preference for rhDHFR.4 The elucidation of the origin of these small differences in terms of specific structural differences between the enzymes and resulting differences in molecular interactions represents an interesting and challenging problem which lends itself to computational study using modern-day molecular simulation techniques.5 The structurally-aligned primary sequences of clDHFR and rhDHFR are compared below for sections that contain most of the important active-site residues (within ca. 8Å of the pterin center): As may be seen, the sequences differ in codons 31, 32 and 39 (Y= tyrosine, F = phenylalanine, K = lysine, R = arginine, S = serine, T = threonine). Codon 31 is of particular interest as the side chain closes over the active site in both rhDHFR and rhDHFR, thereby helping to eliminate solvent from the reaction. We have used computer simulation methods to calculate the free energy change for the transformation Y → F in order to gain some understanding of the species-dependent differences in enzyme activity.
KeywordsFree Energy Change Free Energy Difference Free Energy Calculation Chemical Mutation Thermodynamic Cycle
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