Stereochemistry of phosphoenolpyruvate carboxylation catalyzed by phosphoenolpyruvate carboxykinase

Hwang, Seung Hee; Nowak, Thomas

doi:10.1021/bi00367a037

Cited by 17 publications

(15 citation statements)

References 25 publications

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“…The ultimate products were identified as 3-fluorolactate, (2R,3R)-3-fluoromalate, and (2R,3S)-3-fluoromalate by comparison to authentic samples (Figure 1). The assignments are consistent with those of Keck et al (1980) and Hwang and Nowak (1986).…”

Section: Resultssupporting

confidence: 91%

“…In addition, the ratio of (2R,3R)-3-fluoromalate to (2R,3S)-3-fluoromalate was constant, indicating that reduction of F-OAA was rapid relative to racemization. Goldstein et al (1978) and Hwang and Nowak (1986) reported that racemic 3-fluorooxalacetate was converted by MDH to a mixture of (2R,3R)-3-fluoromalate and (2R,3S)-3-fluoromalate without significant stereoselectivity." While (a-F-PEP is only carboxylated 3% of the time, (Z)-Cl-PEP is reported to undergo 25% carboxylation and 75% hydrolysis (Liu et al, 1990).…”

Section: Resultsmentioning

confidence: 99%

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Mechanistic studies of phosphoenolpyruvate carboxylase from Zea mays with (Z)- and (E)-3-fluorophosphoenolpyruvate as substrates

Janc

Urbauer²,

O’Leary³

et al. 1992

Biochemistry

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The catalytic mechanism of phosphoenolpyruvate (PEP) carboxylase from Zea mays has been studied using (Z)- and (E)-3-fluorophosphoenolpyruvate (F-PEP) as substrates. Both (Z)- and (E)-F-PEP partition between carboxylation to produce 3-fluorooxalacetate and hydrolysis to produce 3-fluoropyruvate. Carboxylation accounts for 3% of the reaction observed with (Z)-F-PEP, resulting in the formation of (R)-3-fluorooxalacetate, and for 86% of the reaction of (E)-F-PEP forming (S)-3-fluorooxalacetate. Carboxylation of F-PEP occurs on the 2-re face, which corresponds to the 2-si face of PEP. The partitioning of F-PEP between carboxylation and hydrolysis is insensitive to pH but varies with metal ion. Use of 18O-labeled bicarbonate produces phosphate that is multiply labeled with 18O; in addition, 18O is also incorporated into residual (Z)- and (E)-F-PEP. The 13(V/K) isotope effect on the carboxylation of F-PEP catalyzed by PEP carboxylase at pH 8.0, 25 degrees C, is 1.049 +/- 0.003 for (Z)-F-PEP and 1.009 +/- 0.006 for (E)-F-PEP. These results are consistent with a mechanism in which carboxylation of PEP occurs via attack of the enolate of pyruvate on CO2 rather than carboxy phosphate. In this mechanism phosphorylation of bicarbonate to give carboxy phosphate and decarboxylation of the latter are reversible steps. An irreversible step, however, precedes partitioning between carboxylation to give oxalacetate and release of CO2, which results in hydrolysis of PEP.

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Section: Resultssupporting

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Mechanistic studies of phosphoenolpyruvate carboxylase from Zea mays with (Z)- and (E)-3-fluorophosphoenolpyruvate as substrates

Janc

Urbauer²,

O’Leary³

et al. 1992

Biochemistry

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“…8A) and that their active site cysteines act as the acid/base catalyst. The occurrence of neutral residues for the second group of enzymes agrees with their documented 2-si-stereoreactivity: PK (63,64), PEPC (65), DAHPS (66), EPSPS (67), MurA (68), and PEPCK (69). The potential catalytic residues of these enzymes were localized in these superimposed structures by comparison with the Cys-115 of MurA (Fig.…”

Section: Cys-502 Counterparts In Homologous Ei and Insupporting

confidence: 73%

Mechanism-based Inhibition of Enzyme I of the Escherichia coli Phosphotransferase System

Garcia-Alles¹,

Flükiger²,

Hewel³

et al. 2002

Journal of Biological Chemistry

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The bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) 1 catalyzes the uptake and phosphorylation of carbohydrates (1). It is further involved in signal transduction (2), e.g. catabolite repression (3), chemotaxis (4), and allosteric regulation of metabolic enzymes and transporters in response to the availability of carbohydrates (5, 6). The PTS is widely distributed in eubacteria and absent from eukaryotes. It generally consists of two central phosphoryl carriers, EI and HPr, and depending on the species, between 1 and 20 peripheral phosphotransferases, the integral membrane components that recognize and transport hexoses, hexitols, and disaccharides. EI and HPr are the proteins at the top of this divergent phosphorylation cascade (7). EI is a 64-kDa two-domain protein (8, 9). In the aminoterminal domain (EIN, residues 1-250) is located His-189, the amino acid transiently phosphorylated by PEP. The threedimensional structure of EIN has been elucidated (10, 11) and its mode of interaction with HPr characterized by NMR spectroscopy (12, 13). It is composed of a HPr binding ␣-helical subdomain and an ␣/␤ subdomain, structurally similar to the phosphohistidine swivel domain of pyruvate phosphate dikinase (PPDK). Phospho-EIN transfers the phosphoryl group to HPr, but it requires the presence of the carboxyl-terminal domain (EIC) to become phosphorylated by PEP (8,14). EIC contains the PEP binding site (15) and plays a crucial role in dimerization (16,17). It also confers species and subunit specificity during the phosphoryl transfer to the next phosphocarrier protein (14). EIC is proteolytically unstable and flexible. Its structure is not yet known. However, the amino acid sequence similarity between EIC and the PEP binding domain of PPDK of Clostridium symbiosum points toward similar fold and structure. There is also sequence similarity with PEP synthase (18).The mode of action of EI and the way its activity is controlled are not yet fully understood. The protein dimerizes in a temperature-dependent manner (19). The dissociation constant has been reported to shift from 20 M at 6°C to 0.9 M at 30°C. The presence of divalent cations (Mg 2ϩ or Mn 2ϩ ) and PEP also affects the equilibrium and the association and dissociation rate constants (20,21). The association rate constant is 2-3 orders of magnitude slower than in other dimeric proteins, * This work was supported by Grant 31-45838.95 from the Swiss National Science Foundation and a fellowship from the Secretaría de Estado de Educación y Universidades, Spain (to L. F. G.-A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.‡ To whom correspondence should be addressed. Tel.: 41-0-31-631-3792; Fax: 41-0-31-631-4887; E-mail:garcia@ibc.unibe.ch.1 The abbreviations used are: PTS, phosphoenolpyruvate:glucose phosphotransferase system; Z-and E-Cl-PEP, (Z)-3-and (E)-3-chlorophos...

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“…Most of the pseudosubstrates or competitive inhibitors discovered so far differed from PEP by the presence of substitutions distal to the phosphate group (position C-3, similar to compounds 1b-e, Scheme 1) [5][6][7]. Some of these compounds turned out to be crucial in mechanistic studies of PEP-utilizing enzymes, for instance in the establishment of the stereochemical course of enzymatic processes mediated by enzyme I of the PTS [8], UDP-GlcNAc enolpyruvyl transferase and 5-enolpyruvyl-shikimate-3-phosphate synthase [9], 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase [10], pyruvate kinase [11], KDOP synthase [12], enolase [13], PEP carboxykinase [14], and PEP carboxylase [15]. A representative example is the study of UDP-GlcNAc enolpyruvyl transferase, and 5-enolpyruvyl-shikimate-3-phosphate synthase, with (Z)-F-PEP (1b), an analogue that allowed the isolation and characterization of stable fluoro analogues of the otherwise unstable tetrahedral intermediate of the normal reaction [1].…”

mentioning

confidence: 99%

Synthesis of phosphoenol pyruvate (PEP) analogues and evaluation as inhibitors of PEP-utilizing enzymes

Garcia-Alles¹,

Erni²

2002

Eur J Biochem

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The synthesis of 10 new phosphoenolpyruvate (PEP) analogues with modifications in the phosphate and the carboxylate function is described. Included are two potential irreversible inhibitors of PEP-utilizing enzymes. One incorporates a reactive chloromethylphosphonate function replacing the phosphate group of PEP. The second contains a chloromethyl group substituting for the carboxylate function of PEP. An improved procedure for the preparation of the known (Z)- and (E)-3-chloro-PEP is also given. The isomers were obtained as a 4 : 1 mixture, resolved by anion-exchange chromatography after the last reaction step. The stereochemistry of the two isomers was unequivocally assigned from the (3)J(H-C) coupling constants between the carboxylate carbons and the vinyl protons. All of these and other known PEP-analogues were tested as reversible and irreversible inhibitors of Mg2+- and Mn2+- activated PEP-utilizing enzymes: enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), pyruvate kinase, PEP carboxylase and enolase. Without exception, the most potent inhibitors were those with substitution of a vinyl proton. Modification of the phosphate and the carboxylate groups resulted in less effective compounds. Enzyme I was the least tolerant to such modifications. Among the carboxylate-modified analogues, only those replaced by a negatively charged group inhibited pyruvate kinase and enolase. Remarkably, the activity of PEP carboxylase was stimulated by derivatives with neutral groups at this position in the presence of Mg2+, but not with Mn2+. For the irreversible inhibition of these enzymes, (Z)-3-Cl-PEP was found to be a very fast-acting and efficient suicide inhibitor of enzyme I (t(1/2) = 0.7 min).

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Stereochemistry of phosphoenolpyruvate carboxylation catalyzed by phosphoenolpyruvate carboxykinase

Cited by 17 publications

References 25 publications

Mechanistic studies of phosphoenolpyruvate carboxylase from Zea mays with (Z)- and (E)-3-fluorophosphoenolpyruvate as substrates

Mechanistic studies of phosphoenolpyruvate carboxylase from Zea mays with (Z)- and (E)-3-fluorophosphoenolpyruvate as substrates

Mechanism-based Inhibition of Enzyme I of the Escherichia coli Phosphotransferase System

Synthesis of phosphoenol pyruvate (PEP) analogues and evaluation as inhibitors of PEP-utilizing enzymes

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