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Peroxide oxidation of indole to oxindole by chloroperoxidase catalysis.

ABSTRACT: In the presence of chloroperoxidase, indole was oxidized by H2O2 to give oxindole as the major product. Under most conditions oxindole was the only product formed, and under optimal conditions the conversion was quantitative. This reaction displayed maximal activity at pH 4.6, although appreciable activity was observed throughout the entire pH range investigated, namely pH 2.5-6.0. Enzyme saturation by indole could not be demonstrated, up to the limit of indole solubility in the buffer. The oxidation kinetics were first-order with respect to indole up to 8 mM, which was the highest concentration of indole that could be investigated. On the other hand, 2-methylindole was not affected by H2O2 and chloroperoxidase, but was a strong inhibitor of indole oxidation. The isomer 1-methylindole was a poor substrate for chloroperoxidase oxidation, and a weak inhibitor of indole oxidation. These results suggest the possibility that chloroperoxidase oxidation of the carbon atom adjacent to the nitrogen atom in part results from hydrogen-bonding of the substrate N-H group to the enzyme active site.

SUBMITTER: Corbett MD

PROVIDER: S-EPMC1161555 | biostudies-other | 1979 Nov

REPOSITORIES: biostudies-other

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Project description:Substrate binding, product release, and likely chemical catalysis in the tryptophan biosynthetic enzyme indole-3-glycerol phosphate synthase (IGPS) are dependent on the structural dynamics of the ?1?1 active-site loop. Statistical coupling analysis and molecular dynamic simulations had previously indicated that covarying residues in the ?1?1 and ?2?2 loops, corresponding to Arg54 and Asn90, respectively, in the Sulfolobus sulfataricus enzyme (ssIGPS), are likely important for coordinating functional motions of these loops. To test this hypothesis, we characterized site mutants at these positions for changes in catalytic function, protein stability and structural dynamics for the thermophilic ssIGPS enzyme. Although there were only modest changes in the overall steady-state kinetic parameters, solvent viscosity and solvent deuterium kinetic isotope effects indicated that these amino acid substitutions change the identity of the rate-determining step across multiple temperatures. Surprisingly, the N90A substitution had a dramatic effect on the general acid/base catalysis of the dehydration step, as indicated by the loss of the descending limb in the pH rate profile, which we had previously assigned to Lys53 on the ?1?1 loop. These changes in enzyme function are accompanied with a quenching of ps-ns and µs-ms timescale motions in the ?1?1 loop as measured by nuclear magnetic resonance studies. Altogether, our studies provide structural, dynamic and functional rationales for the coevolution of residues on the ?1?1 and ?2?2 loops, and highlight the multiple roles that the ?1?1 loop plays in IGPS catalysis. Thus, substitution of covarying residues in the active-site ?1?1 and ?2?2 loops of indole-3-glycerol phosphate synthase results in functional, structural, and dynamic changes, highlighting the multiple roles that the ?1?1 loop plays in enzyme catalysis and the importance of regulating the structural dynamics of this loop through noncovalent interactions with nearby structural elements.

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Peroxide oxidation of indole to oxindole by chloroperoxidase catalysis.

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