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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)...
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| Published in: | Biochemistry (Easton) 2004-03, Vol.43 (9), p.2524-2532 |
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| Main Authors: | , |
| Format: | Article |
| Language: | English |
| Subjects: | |
| Citations: | Items that this one cites Items that cite this one |
| Online Access: | Get full text |
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| Summary: | 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 π 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 Å-2 at 25 °C. Free energy changes accompanying binding of hydroxamate derivatives also correlated well with π 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 Å-2 at 25 °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 μM) that was reduced relative to that observed for benzohydroxamate and that was in accord with the ∼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. |
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| ISSN: | 0006-2960 1520-4995 |
| DOI: | 10.1021/bi036207x |