Inactivation of Mandelate Racemase by 3-Hydroxypyruvate Reveals a Potential Mechanistic Link between Enzyme Superfamilies

Authors

Mitesh Nagar, Dalhousie University
Brittney N. Wyatt, Marquette University
Martin St. Maurice, Marquette UniversityFollow
Stephen L. Bearne, Dalhousie University

Document Type

Article

Language

eng

Format of Original

11 p.

Publication Date

5-5-2015

Publisher

American Chemical Society

Source Publication

Biochemistry

Source ISSN

0006-2960

Original Item ID

DOI: 10.1021/acs.biochem.5b00221, PubMed Central: PMID: 25844917

Abstract

Mandelate racemase (MR), a member of the enolase superfamily, catalyzes the Mg²⁺-dependent interconversion of the enantiomers of mandelate. Several α-keto acids are modest competitive inhibitors of MR [e.g., mesoxalate (K_i = 1.8 ± 0.3 mM) and 3-fluoropyruvate (K_i = 1.3 ± 0.1 mM)], but, surprisingly, 3-hydroxypyruvate (3-HP) is an irreversible, time-dependent inhibitor (k_inact/K_I = 83 ± 8 M^–1 s^–1). Protection from inactivation by the competitive inhibitor benzohydroxamate, trypsinolysis and electrospray ionization tandem mass spectrometry analyses, and X-ray crystallographic studies reveal that 3-HP undergoes Schiff-base formation with Lys 166 at the active site, followed by formation of an aldehyde/enol(ate) adduct. Such a reaction is unprecedented in the enolase superfamily and may be a relic of an activity possessed by a promiscuous progenitor enzyme. The ability of MR to form and deprotonate a Schiff-base intermediate furnishes a previously unrecognized mechanistic link to other α/β-barrel enzymes utilizing Schiff-base chemistry and is in accord with the sequence- and structure-based hypothesis that members of the metal-dependent enolase superfamily and the Schiff-base-forming N-acetylneuraminate lyase superfamily and aldolases share a common ancestor.

Comments

Accepted version. Biochemistry, Vol. 54, No. 17 (May 5, 2015): 2747-2757. DOI. © 2015 American Chemical Society. Used with permission.

Recommended Citation

Nagar, Mitesh; Wyatt, Brittney N.; St. Maurice, Martin; and Bearne, Stephen L., "Inactivation of Mandelate Racemase by 3-Hydroxypyruvate Reveals a Potential Mechanistic Link between Enzyme Superfamilies" (2015). Biological Sciences Faculty Research and Publications. 520.
https://epublications.marquette.edu/bio_fac/520