title: Inactivation of Mandelate Racemase by 3-Hydroxypyruvate Reveals a Potential Mechanistic Link between Enzyme Superfamilies.
abstract: Mandelate racemase (MR), a member of the enolase superfamily, catalyzes the Mg(2+)-dependent interconversion of the enantiomers of mandelate. Several α-keto acids are modest competitive inhibitors of MR [e.g., mesoxalate (Ki = 1.8 ± 0.3 mM) and 3-fluoropyruvate (Ki = 1.3 ± 0.1 mM)], but, surprisingly, 3-hydroxypyruvate (3-HP) is an irreversible, time-dependent inhibitor (kinact/KI = 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.
authors.affiliation: †Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS B3H 4R2, Canada.
authors.forename: Mitesh
authors.initials: M
authors.lastname: Nagar
authors.affiliation: ‡Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin 53201-1881, United States.
authors.forename: Brittney N
authors.initials: BN
authors.lastname: Wyatt
authors.affiliation: ‡Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin 53201-1881, United States.
authors.forename: Martin
authors.initials: M
authors.lastname: St Maurice
authors.affiliation: †Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS B3H 4R2, Canada.|§Department of Chemistry, Dalhousie University, Halifax, NS B3H 4R2, Canada.
authors.forename: Stephen L
authors.initials: SL
authors.lastname: Bearne
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D018360:Crystallography, X-Ray; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011773:Pyruvates / Q000494:pharmacology*; D019998:Racemases and Epimerases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D012545:Schiff Bases / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013329:Structure-Activity Relationship; D053719:Tandem Mass Spectrometry
chemical_list: D017931:DNA Primers; D004791:Enzyme Inhibitors; D011773:Pyruvates; D012545:Schiff Bases; C012375:hydroxypyruvic acid; D019998:Racemases and Epimerases; C021540:mandelate racemase