期刊
BIOCHEMISTRY
卷 54, 期 17, 页码 2747-2757出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.biochem.5b00221
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资金
- Natural Sciences and Engineering Research Council (NSERC) of Canada
- National Science Foundation [DBI-0959442]
- U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences [DE-AC02-06CH11357]
- Michigan Economic Development Corp.
- Michigan Technology Tri-Corridor [085P1000817]
- U.S. Department of Education
Mande late racemase (MR), a member of the enolase superfamily, catalyzes the Mg2+-dependent interconversion of the enantiomers of mandelate. Several alpha-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 alpha/beta-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.
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