Reaction intermediate analogues for mandelate racemase: interaction between Asn 197 and the alpha-hydroxyl of the substrate promotes catalysis.
Mandelate racemase (MR) catalyzes the interconversion of the enantiomers of mandelic acid, stabilizing the altered substrate in the transition state by 26 kcal/mol relative to the substrate in the ground state. To understand the origins of this binding discrimination, carboxylate-, phosphonate-, and hydroxamate-containing substrate and intermediate analogues were examined for their ability to inhibit MR. Comparison of the competitive inhibition constants revealed that an alpha-hydroxyl function is required for recognition of the ligand as an intermediate analogue. Two intermediate analogues, alpha-hydroxybenzylphosphonate (alpha-HBP) and benzohydroxamate, were bound with affinities approximately 100-fold greater than that observed for the substrate. Furthermore, MR bound alpha-HBP enantioselectively, displaying a 35-fold higher affinity for the (S)-enantiomer relative to the (R)-enantiomer. In the X-ray structure of mandelate racemase [Landro, J. A., Gerlt, J. A., Kozarich, J. W., Koo, C. W., Shah, V. J., Kenyon, G. L., Neidhart, D. J., Fujita, J., and Petsko, G. A. (1994) Biochemistry 33, 635-643], the alpha-hydroxyl function of the competitive inhibitor (S)-atrolactate is within hydrogen bonding distance of Asn 197. To demonstrate the importance of the alpha-hydroxyl function in intermediate binding, the N197A mutant was constructed. The values of k(cat) for N197A were reduced 30-fold for (R)-mandelate and 179-fold for (S)-mandelate relative to wild-type MR; the values of k(cat)/K(m) were reduced 208-fold for (R)-mandelate and 556-fold for (S)-mandelate. N197A shows only a 3.5-fold reduction in its affinity for the substrate analogue (R)-atrolactate but a 51- and 18-fold reduction in affinity for alpha-HBP and benzohydroxamate, respectively. Thus, interaction between Asn 197 and the substrate's alpha-hydroxyl function provides approximately 3.5 kcal/mol of transition-state stabilization free energy to differentially stabilize the transition state relative to the ground state.
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Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, B3H 4H7 Canada.
Bearne
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D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D002264:Carboxylic Acids / Q000737:chemistry; D002384:Catalysis; D007700:Kinetics; D008333:Mandelic Acids / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D063065:Organophosphonates / Q000737:chemistry; D011522:Protons; D016958:Pseudomonas putida / Q000201:enzymology; D019998:Racemases and Epimerases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D055672:Static Electricity; D013237:Stereoisomerism; D013379:Substrate Specificity
D002264:Carboxylic Acids; D008333:Mandelic Acids; D063065:Organophosphonates; D011522:Protons; D001216:Asparagine; D019998:Racemases and Epimerases; C021540:mandelate racemase; C037938:mandelic acid; D000409:Alanine