Mechanism of the reaction catalyzed by mandelate racemase. 3. Asymmetry in reactions catalyzed by the H297N mutant

Landro, James A.; Kallarakal, Abraham T.; Ransom, Stephen C.; Gerlt, J.A.; Kozarich, John W.; Neidhart, David J.; Kenyon, George L.

doi:10.1021/bi00102a020

Cited by 87 publications

(124 citation statements)

References 16 publications

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“…Two catalytic bases, K166 and H297, are employed to accomplish this task (21,26,27). From their positions in the active site, it seems that D270 assists H297 in proton abstraction, and that together they form a histidine͞aspartate dyad that can more properly be considered the second catalytic base.…”

Section: Resultsmentioning

confidence: 99%

Evolution of an enzyme active site: The structure of a new crystal form of muconate lactonizing enzyme compared with mandelate racemase and enolase

Hasson

Schlichting

Moulai

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

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Muconate lactonizing enzyme (MLE), a component of the ␤-ketoadipate pathway of Pseudomonas putida, is a member of a family of related enzymes (the ''enolase superfamily'') that catalyze the abstraction of the ␣-proton of a carboxylic acid in the context of different overall reactions. New untwinned crystal forms of MLE were obtained, one of which diffracts to better than 2.0-Å resolution. The packing of the octameric enzyme in this crystal form is unusual, because the asymmetric unit contains three subunits. The structure of MLE presented here contains no bound metal ion, but is very similar to a recently determined Mn 2؉ -bound structure. Thus, absence of the metal ion does not perturb the structure of the active site. The structures of enolase, mandelate racemase, and MLE were superimposed. A comparison of metal ligands suggests that enolase may retain some characteristics of the ancestor of this enzyme family. Comparison of other residues involved in catalysis indicates two unusual patterns of conservation: (i) that the position of catalytic atoms remains constant, although the residues that contain them are located at different points in the protein fold; and (ii) that the positions of catalytic residues in the protein scaffold are conserved, whereas their identities and roles in catalysis vary.

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Section: Resultsmentioning

confidence: 99%

Evolution of an enzyme active site: The structure of a new crystal form of muconate lactonizing enzyme compared with mandelate racemase and enolase

Hasson

Schlichting

Moulai

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

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“…, resolution with (ϩ)-ephedrine (22)]; 2-2 H-p-substituted mandelic acids were prepared using the H297N mutant of mandelate racemase (23) and were a gift from John A. Gerlt (University of Illinois).…”

Section: Methodsmentioning

confidence: 99%

On the reaction mechanism of l -lactate oxidase: Quantitative structure-activity analysis of the reaction with para -substituted l -mandelates

Yorita

Janko

Aki

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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The rate constants for reduction of the f lavoenzyme, L-lactate oxidase, and a mutant (in which alanine 95 is replaced by glycine), by a series of para-substituted mandelates, in both the 2-1 H-and 2-2 H-forms, have been measured by rapid reaction spectrophotometry. In all cases, significant isotope effects ( 1 H͞ 2 H ‫؍‬ 3-7) on the rate constants of f lavin reduction were found, indicating that f lavin reduction is a direct measure of ␣-C-H bond breakage. The rate constants show only a small inf luence of the electronic characteristics of the substituents, but show a good correlation when combined with some substituent volume parameters. A surprisingly good correlation is found with the molecular mass of the substrate. The results are compatible with any mechanism in which there is little development of charge in the transition state. This could be a transfer of hydride to the f lavin N(5) position or a synchronous mechanism in which the ␣-C-H is formally abstracted as a H ؉ while the resulting charge is simultaneously neutralized by another event.L-Lactate oxidase is a newly studied member of the family of FMN-containing enzymes that catalyze the oxidation of ␣-hydroxyacids, comprising glycolate oxidase (EC 1.1.3.1), Llactate oxidase, L-lactate monooxygenase (EC 1.13.12.4), flavocytochrome b 2 (EC 1.1.2.3), long-chain ␣-hydroxyacid oxidase (EC 1.1.3.15), and L-mandelate dehydrogenase. A considerable amount of mechanistic work has been carried out with L-lactate monooxygenase and with flavocytochrome b 2 (for reviews, see refs. 1 and 2), which has been interpreted as supporting a carbanion mechanism, in which the substrate ␣-proton is abstracted by an active site base and the electrons from the resulting substrate carbanion are transferred to the flavin. The crystal structures of two of the family members, glycolate oxidase (3) and flavocytochrome b 2 (4), have been solved and show an arrangement of conserved protein residues surrounding the FMN prosthetic group and substrate that are consistent with such a carbanion mechanism. In these structures, the substrate is positioned on the si-face of the flavin with the carboxylate in ionic interaction with arginine residues and stabilized by H-bond interaction with a tyrosine residue (5). A conserved histidine residue is located so that the ␣-carbon of the substrate would be positioned between the flavin N(5) and the histidine N1. The histidine residue therefore has been envisaged as the active site base responsible for abstracting the proton from the ␣-position of the substrate, with concomitant attack of the highly nucleophilic carbanion on flavin N(5).While the crystal structures of only two of the family members are available so far, the amino acid sequences of all the enzymes are known: glycolate oxidase (6, 7), flavocytochrome b 2 (8), lactate monooxygenase (9), lactate oxidase (10), long chain ␣-hydroxyacid oxidase (11), and mandelate dehydrogenase (12, 13). All enzymes of the group show considerable homology, and all have the strictly conserved set o...

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“…MR catalyzes a 1,1-proton transfer reaction in which the enantiomers of mandelate are equilibrated by a two-base mechanism [4]: Lys 166 is the (S)-specific base that mediates proton transfers to/from (S)-mandelate [5]; His 297, hydrogen bonded to Asp 270 in a His-Asp dyad, is the (R)-specific base [6]. MR catalyzes a 1,1-proton transfer reaction in which the enantiomers of mandelate are equilibrated by a two-base mechanism [4]: Lys 166 is the (S)-specific base that mediates proton transfers to/from (S)-mandelate [5]; His 297, hydrogen bonded to Asp 270 in a His-Asp dyad, is the (R)-specific base [6].…”

Section: The Kinetic Problems Associated With Proton Abstraction Frommentioning

confidence: 99%

“…So, for example, in the case of the mandelate racemase-catalyzed reaction, for which the value of the pK a of mandelate anion is 29 [1] and the value of the pK a of Lys 166, the (S)-specific base, is 6 [6], the value of k cat would no larger than 6:2 Â 10 À11 if the enolate anion intermediate were not stabilized in the active site; this value is @10 13 -fold less than the observed value for the k cat , 500 s À1 . Recall that an enolate anion is necessarily on the reaction coordinate, so the value of DG o must be reduced for the enolate anion to be kinetically competent irrespective of whether DG z int can be reduced.…”

Section: 22mentioning

confidence: 99%

Enzymatic Catalysis of Proton Transfer at Carbon Atoms

Gerlt

2006

Hydrogen‐Transfer Reactions

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Mechanism of the reaction catalyzed by mandelate racemase. 3. Asymmetry in reactions catalyzed by the H297N mutant

Cited by 87 publications

References 16 publications

Evolution of an enzyme active site: The structure of a new crystal form of muconate lactonizing enzyme compared with mandelate racemase and enolase

Evolution of an enzyme active site: The structure of a new crystal form of muconate lactonizing enzyme compared with mandelate racemase and enolase

On the reaction mechanism of l -lactate oxidase: Quantitative structure-activity analysis of the reaction with para -substituted l -mandelates

Enzymatic Catalysis of Proton Transfer at Carbon Atoms

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