Hydrophobic nature of the active site of mandelate racemase.
Mandelate racemase (EC 5.1.2.2) from Pseudomonas putida catalyzes the interconversion of the two enantiomers of mandelic acid with remarkable proficiency, stabilizing the altered substrate in the transition state by approximately 26 kcal/mol. We have used a series of substrate analogues (glycolates) and intermediate analogues (hydroxamates) to evaluate the contribution of the hydrophobic cavity within the enzyme's active site to ligand binding. Free energy changes accompanying binding of glycolate derivatives correlated well with the hydrophobic substituent constant pi and the van der Waals surface areas of the ligands. The observed dependence of the apparent binding free energy on surface area of the ligand was -30 +/- 5 cal mol(-1) A(-2) at 25 degrees C. Free energy changes accompanying binding of hydroxamate derivatives also correlated well with pi values and the van der Waals surface areas of the ligands, giving a slightly greater free energy dependence equal to -41 +/- 3 cal mol(-1) A(-2) at 25 degrees C. Surprisingly, mandelate racemase exhibited a binding affinity for the intermediate analogue benzohydroxamate that was 2 orders of magnitude greater than that predicted solely on the basis of hydrophobic interactions. This suggests that there are additional specific interactions that stabilize the altered substrate in the transition state. Mandelate racemase was competitively inhibited by (R,S)-1-naphthylglycolate (apparent K(i) = 1.9 +/- 0.1 mM) and (R,S)-2-naphthylglycolate (apparent K(i) = 0.52 +/- 0.03 mM), demonstrating the plasticity of the hydrophobic pocket. Both (R)- (K(m) = 0.46 +/- 0.06 mM, k(cat) = 33 +/- 1 s(-1)) and (S)-2-naphthylglycolate (K(m) = 0.41 +/- 0.03 mM, k(cat) = 25 +/- 1 s(-1)) were shown to be alternative substrates for mandelate racemase. These kinetic results demonstrate that no major steric restrictions are imposed on the binding of this bulkier substrate in the ground state but that steric factors appear to impair transition state/intermediate stabilization. 2-Naphthohydroxamate was identified as a competitive inhibitor of mandelate racemase, binding with an affinity (K(i) = 57 +/- 18 microM) that was reduced relative to that observed for benzohydroxamate and that was in accord with the approximately 10-fold reduction in the value of k(cat)/K(m) for the racemization of 2-naphthylglycolate. These findings indicate that, for mandelate racemase, steric constraints within the hydrophobic cavity of the enzyme-intermediate complex are more stringent than those in the enzyme-substrate complex.
Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada.
Martin
M
St  Maurice
Stephen L
SL
Bearne
D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D006016:Glycolates / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D006877:Hydroxamic Acids / Q000737:chemistry; D008024:Ligands; D008333:Mandelic Acids / Q000737:chemistry; D011485:Protein Binding; D016958:Pseudomonas putida / Q000201:enzymology; D019998:Racemases and Epimerases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D013237:Stereoisomerism; D013379:Substrate Specificity; D013499:Surface Properties; D013816:Thermodynamics
D006016:Glycolates; D006877:Hydroxamic Acids; D008024:Ligands; D008333:Mandelic Acids; D019998:Racemases and Epimerases; C021540:mandelate racemase; C037938:mandelic acid