Inhibition of isocitrate lyase by 3-nitropropionate, a reaction-intermediate analog

Schloss, John V.; Cleland, W. W.

doi:10.1021/bi00261a035

Cited by 92 publications

(77 citation statements)

References 34 publications

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“…On the basis of the work reported here, it is hypothesized that the mechanism of PrpB action is similar to that of ICL based on the similarities in structure of substrates, and in similarities between the active site loop region between ICL and 2-MICL, i.e., the K(R/ K)CGH motif. Both ICL and E. coli PrpB can be inactivated by treatment with cysteine-modifying agents (7,31). Our data indicate that residue C123 is critical to catalysis.…”

Section: Discussionmentioning

confidence: 81%

Residues C123 and D58 of the 2-Methylisocitrate Lyase (PrpB) Enzyme ofSalmonella entericaAre Essential for Catalysis

Grimek¹,

Holden

Rayment

et al. 2003

J Bacteriol

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The prpB gene of Salmonella enterica serovar Typhimurium LT2 encodes a protein with 2-methylisocitrate (2-MIC) lyase activity, which cleaves 2-MIC into pyruvate and succinate during the conversion of propionate to pyruvate via the 2-methylcitric acid cycle. This paper reports the isolation and kinetic characterization of wild-type and five mutant PrpB proteins. Wild-type PrpB protein had a molecular mass of approximately 32 kDa per subunit, and the biologically active enzyme was comprised of four subunits. Optimal 2-MIC lyase activity was measured at pH 7.5 and 50°C, and the reaction required Mg 2؉ ions; equimolar concentrations of Mn 2؉ ions were a poor substitute for Mg 2؉ (28% specific activity). Dithiothreitol (DTT) or reduced glutathione (GSH) was required for optimal activity; the role of DTT or GSH was apparently not to reduce disulfide bonds, since the disulfide-specific reducing agent Tris(2-carboxyethyl)phosphine hydrochloride failed to substitute for DTT or GSH. The K m of PrpB for 2-MIC was measured at 19 M, with a k cat of 105 s ؊1 . Mutations in the prpB gene were introduced by site-directed mutagenesis based on the active-site residues deemed important for catalysis in the closely related phosphoenolpyruvate mutase and isocitrate lyase enzymes. Residues D58, K121, C123, and H125 of PrpB were changed to alanine, and residue R122 was changed to lysine. Nondenaturing polyacrylamide gel electrophoresis indicated that all mutant PrpB proteins retained the same oligomeric state of the wild-type enzyme, which is known to form tetramers. The PrpB K121A , PrpB H125A , and PrpB R122K mutant proteins formed enzymes that had 1,050-, 750-, and 2-fold decreases in k cat for 2-MIC lyase activity, respectively. The PrpB D58A and PrpB C123A proteins formed tetramers that displayed no detectable 2-MIC lyase activity indicating that both of these residues are essential for catalysis. Based on the proposed mechanism of the closely related isocitrate lyases, PrpB residue C123 is proposed to serve as the active site base, and residue D58 is critical for the coordination of a required Mg 2؉ ion.Propionate catabolism in Salmonella enterica serovar Typhimurium LT2 (herein referred to as serovar Typhimurium) occurs via the 2-methylcitric acid (2-MC) cycle (Fig. 1) (18). The 2-MC cycle of propionate metabolism was first identified in the yeast Yarrowia lipolytica and several years later was found to exist in prokaryotes (8,18,36,37). The prokaryotic enzymes of this pathway have been characterized. PrpE is the propionyl coenzyme A synthetase (14, 17), PrpC is the 2-MC synthase (18, 39), PrpD is the 2-MC dehydratase (8, 15), and PrpB is the 2-methylisocitrate (2-MIC) lyase (7,18). This work focuses on the serovar Typhimurium PrpB enzyme, which catalyzes the cleavage of 2-MIC into pyruvate and succinate (15).Comparisons of the amino acid sequence of the PrpB protein to other protein sequences identified it as a homolog of carboxyphosphonoenolpyruvate (CPEP) mutase, phosphoenolpyruvate (PEP) mutase, and isocitrate lyases (ICL) ...

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Section: Discussionmentioning

confidence: 81%

Residues C123 and D58 of the 2-Methylisocitrate Lyase (PrpB) Enzyme ofSalmonella entericaAre Essential for Catalysis

Grimek¹,

Holden

Rayment

et al. 2003

J Bacteriol

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“…The catalytic mechanism of Mtb ICL derived from both structural data (10) and kinetic analysis (13)(14)(15) is depicted in Fig. 1.…”

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confidence: 99%

Mechanism-based inactivator of isocitrate lyases 1 and 2 fromMycobacterium tuberculosis

Pham

Murkin

Moynihan

et al. 2017

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

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Isocitrate lyase (ICL, types 1 and 2) is the first enzyme of the glyoxylate shunt, an essential pathway for Mycobacterium tuberculosis (Mtb) during the persistent phase of human TB infection. Here, we report 2-vinyl-D-isocitrate (2-VIC) as a mechanism-based inactivator of Mtb ICL1 and ICL2. The enzyme-catalyzed retro-aldol cleavage of 2-VIC unmasks a Michael substrate, 2-vinylglyoxylate, which then forms a slowly reversible, covalent adduct with the thiolate form of active-site Cys 191 . 2-VIC displayed kinetic properties consistent with covalent, mechanism-based inactivation of ICL1 and ICL2 with high efficiency (partition ratio, <1). Analysis of a complex of ICL1:2-VIC by electrospray ionization mass spectrometry and X-ray crystallography confirmed the formation of the predicted covalent S-homopyruvoyl adduct of the active-site Cys 191 .mechanism-based inactivation | isocitrate lyase | tuberculosis | 2-vinyl isocitrate | covalent adduct

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“…The low value of 8.82 x 102 M-'S-' for invertase and invertase inhibitor association suggests that another rate-determining step controls rate of the association. Slow association of enzymes with large inhibitors has been reported for other systems (5,7,14,18 …”

Section: Discussionmentioning

confidence: 80%

Characteristics of the Inhibition of Potato (Solanum tuberosum) Invertase by an Endogenous Proteinaceous Inhibitor in Potatoes

Bracho

Whitaker²

1990

Plant Physiol.

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Effect of several parameters on inhibition of potato (Solanum tuberosum) invertase by its endogenous proteinaceous inhibitor was determined using homogeneous preparations of both proteins. The inhibitor and invertase formed an inactive complex with an observed association rate constant at pH 4.70 and 37°C of 8.82 x 102 per molar per second and a dissociation rate constant of 3.3 x 10-3 per minute. The inhibitor appeared to bind to invertase in more than one step. Initial interaction (measured by loss of invertase activity) was rapid, relatively weak, readily reversible (K, of 2 x 10-6 molar) and noncompetitive with substrate at pH 4.70. Initial interaction was probably followed by isomerization to a tighter (K, of 6.23 x 10-i molar) complex, which dissociated slowly with a half-time of 3.5 hour. Interaction between enzyme and inhibitor appeared to be of ionic character and essentially pH independent between pH 3.5 and 7.4. Schwimmer et al. (19) first reported evidence for an endogenous proteinaceous inhibitor ofinvertase in potatoes. Pressey (15,16) purified the inhibitor to homogeneity and partially characterized it. Invertase inhibitors have been found also in red beet, sugar beet, and sweet potato (1 1, 17) and in maize endosperm (9).Invertase inhibitors have been implicated in the regulation of invertase activity in several plant tissues. In potato tubers, the inhibitor may play a role in the increase of invertase activity resulting from cold treatment, wounding, or transferring of callus into liquid medium. Some aspects of the interaction between potato invertase and invertase inhibitor using crude preparations of both proteins have been reported by Ewing and McAdoo (6)

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Inhibition of isocitrate lyase by 3-nitropropionate, a reaction-intermediate analog

Cited by 92 publications

References 34 publications

Residues C123 and D58 of the 2-Methylisocitrate Lyase (PrpB) Enzyme ofSalmonella entericaAre Essential for Catalysis

Residues C123 and D58 of the 2-Methylisocitrate Lyase (PrpB) Enzyme ofSalmonella entericaAre Essential for Catalysis

Mechanism-based inactivator of isocitrate lyases 1 and 2 fromMycobacterium tuberculosis

Characteristics of the Inhibition of Potato (Solanum tuberosum) Invertase by an Endogenous Proteinaceous Inhibitor in Potatoes

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