2-Oxo-3-alkynoic acids, universal mechanism-based inactivators of thiamin diphosphate-dependent decarboxylases: synthesis and evidence for potent inactivation of the pyruvate dehydrogenase multienzyme complex.
A new class of compounds, the 2-oxo-3-alkynoic acids with a phenyl substituent at carbon 4 was reported by the authors as potent irreversible and mechanism-based inhibitors of the thiamin diphosphate- (ThDP-) dependent enzyme pyruvate decarboxylase [Chiu, C.-F., & Jordan, F. (1994) J. Org. Chem. 59, 5763-5766]. The method has been successfully extended to the synthesis of the 4-, 5-, and 7-carbon aliphatic members of this family of compounds. These three compounds were then tested on three ThDP-dependent pyruvate decarboxylases: the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) and its E1 (ThDP-dependent) component, pyruvate oxidase (POX, phosphorylating; from Lactobacillus plantarum),and pyruvate decarboxylase (PDC) from Saccharomycescerevisiae. All three enzymes were irreversibly inhibited by the new compounds. The 4-carbon acid is the best substrate-analog inactivator known to date for PDHc, more potent than either fluoropyruvate or bromopyruvate. The following conclusions were drawn from extensive studies with PDHc: (a) The kinetics of inactivation of PDH complexes and of resolved E1 by 2-oxo-3-alkynoic acids is time- and concentration-dependent. (b) The 4-carbon acid has a Ki 2 orders of magnitude stronger than the 5-carbon acid, clearly demonstrating the substrate specificity of PDHc. (c) The rate of inactivation of PDH complexes and of resolved E1 by 2-oxo-3-alkynoic acids is enhanced by the addition of ThDP and MgCl2. (d) Pyruvate completely protects E1 and partially protects PDHc from inactivation by 2-oxo-3-butynoic acid. (e) E1 but not E2-E3 is the target of inactivation by 2-oxo-3-butynoic acid. (f) Inactivation of E1 by 2-oxo-3-butynoic acid is accompanied by modification of 1.3 cysteines/E1 monomer. The order of reactivity with the 4-carbon acid was PDHc > POX > PDC. While the order of reactivity with PDHc and POX was 2-oxo-3-butynoic acid > 2-oxo-3-pentynoic acid > 2-oxo-3-heptynoic acid, the order of reactivity was reversed with PDC.
Brown
A
A
Department of Chemistry, Rutgers, The State University of New Jersey, Newark, New Jersey 07102, USA.
Nemeria
N
N
Yi
J
J
Zhang
D
D
Jordan
W B
WB
Machado
R S
RS
Guest
J R
JR
Jordan
F
F
D001426:Bacterial Proteins / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology*; D005656:Fungal Proteins / Q000378:metabolism; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology; D011767:Pyruvate Decarboxylase / Q000378:metabolism*; D011768:Pyruvate Dehydrogenase Complex / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000378:metabolism*; D011771:Pyruvate Oxidase / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013835:Thiamine Pyrophosphate / Q000378:metabolism*
D001426:Bacterial Proteins; D005231:Fatty Acids, Unsaturated; D005656:Fungal Proteins; D011768:Pyruvate Dehydrogenase Complex; C037086:2-keto-3-butynoic acid; D011771:Pyruvate Oxidase; D011767:Pyruvate Decarboxylase; D013835:Thiamine Pyrophosphate