Fluorine NMR studies on stereochemical aspects of reactions catalyzed by transcarboxylase, pyruvate kinase, and enzyme I

Hoving, Henk; Crysell, Brian; Leadlay, Peter F.

doi:10.1021/bi00343a020

Cited by 17 publications

(14 citation statements)

References 25 publications

(31 reference statements)

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“…By use of 19F NMR analysis (Figure 1 A), our results show that malate dehydrogenase has little or no selectivity and uses both enantiomers of 3-fluorooxalacetate to produce both diastereomers of (2R)-3-fluoromalate. This observation is consistent with the results of Goldstein et al (1978) and has been confirmed by Hoving et al (1985). The…”

Section: Discussionsupporting

confidence: 93%

“…For instance, OH™ is always added from the si-si face of fumarate while H+ is always added from the re-re face of fumarate. More recently, Hoving et al (1985) have studied the stereochemistry of the transcarboxylasecatalyzed carboxylation of 3-fluoropyruvate by using 19F NMR analysis. Their results demonstrated that transcarboxylase is specific for one of the two prochiral hydrogens of fluoropyruvate and converts it to 3(R)-fluorooxalacetate with retention of configuration.…”

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confidence: 99%

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Stereochemistry of phosphoenolpyruvate carboxylation catalyzed by phosphoenolpyruvate carboxykinase

Hwang¹,

Nowak²

1986

Biochemistry

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The stereochemistry of the carboxylation of phosphoenolpyruvate to yield oxalacetate, catalyzed by chicken liver phosphoenolpyruvate carboxykinase and by Ascaris muscle phosphoenolpyruvate carboxykinase, was determined. The substrate (Z)-3-fluorophosphoenolpyruvate was used for the stereochemical analysis. The carboxylation reaction was coupled to malate dehydrogenase to yield 3-fluoromalate, and the stereochemistry of the products was identified by 19F NMR. In separate experiments, the enantiomeric tautomers of 3-fluorooxalacetate were shown to be utilized by malate dehydrogenase to yield (2R,3R)- and (2R,3S)-3-fluoromalate in nearly identical amounts. The products were identified by 19F NMR. When (Z)-3-fluorophosphoenolpyruvate was used as a substrate for phosphoenolpyruvate carboxykinase from avian liver and from Ascaris, and malate dehydrogenase was used to trap the product, only a single diastereomer was observed. This product was shown to be (2R,3R)-3-fluoromalate in each case. The assignments were based on coupling constants taken from Keck et al. [Keck, R., Hess, H., & Rétey, J. (1980) FEBS Lett. 114, 287]. These results indicate that the stereochemistry of carboxylation, catalyzed by chicken phosphoenolpyruvate carboxykinase and by Ascaris phosphoenolpyruvate carboxykinase, is identical and takes place from the si side of the enzyme-bound phosphoenolpyruvate. The carboxylation reaction was run both in H2O and in D2O. No deuterium incorporation into fluoromalate was shown to occur. The product 3-fluorooxalacetate is thus released from phosphoenolpyruvate carboxykinase as the keto form and is reduced more rapidly by reduced nicotinamide adenine dinucleotide with malate dehydrogenase than by the occurrence of tautomerization.

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Section: Discussionsupporting

confidence: 93%

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confidence: 99%

Stereochemistry of phosphoenolpyruvate carboxylation catalyzed by phosphoenolpyruvate carboxykinase

Hwang¹,

Nowak²

1986

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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“…The discrepancy could not be due to decarboxylation of fluoro-oxaloacetate. Efficient reduction of this keto acid by malate dehydrogenase has been previously reported (Hoving et al, 1985;Hwang & Nowak, 1986). The amount of this enzyme added to the assay medium is similar to that used by those authors.…”

Section: Resultsmentioning

confidence: 81%

“…One of the most commonly used analogues of Pyr-P is phosphoenol-3-fluoropyruvate (F-Pyr-P). It has been described as a good substrate for pyruvate kinase (Stubbe & Kenyon, 1972;Blumberg & Stubbe, 1975;Duffy & Nowak, 1984;Hoving et al, 1985), enolase (Stubbe & Kenyon, 1972;Duffy & Nowak, 1984), Pyr-P carboxykinase (Duffy & Nowak, 1984;Hwang & Nowak, 1986), pyruvate,phosphate dikinase (Duffy & Nowak, 1984) and enzyme I of the sugar phosphotransferase system of Escherichia coli (Hoving et al, 1985). Since fluoride is almost isosteric to hydrogen, we decided to test F-Pyr-P as a substrate for Pyr-P carboxylase from maize leaves.…”

Section: Introductionmentioning

confidence: 99%

Carboxylation and dephosphorylation of phosphoenol-3-fluoropyruvate by maize leaf phosphoenolpyruvate carboxylase

Gonzalez

Andreo

1988

Biochemical Journal

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The analogue (Z)-phosphoenol-3-fluoropyruvate [(Z)-3-fluoro-2-(phosphono-oxy)propenoic acid] was tested as substrate of maize leaf phosphoenolpyruvate carboxylase. Studies with NaH14CO3 indicate that the analogue is carboxylated by the enzyme. However, this reaction accounts for only one-tenth of the activity measured by Pi liberation. The rest of the analogue is merely dephosphorylated. This is the first analogue for which both carboxylation and dephosphorylation have been observed.

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Fluorine NMR studies on stereochemical aspects of reactions catalyzed by transcarboxylase, pyruvate kinase, and enzyme I

Cited by 17 publications

References 25 publications

Stereochemistry of phosphoenolpyruvate carboxylation catalyzed by phosphoenolpyruvate carboxykinase

Stereochemistry of phosphoenolpyruvate carboxylation catalyzed by phosphoenolpyruvate carboxykinase

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

Carboxylation and dephosphorylation of phosphoenol-3-fluoropyruvate by maize leaf phosphoenolpyruvate carboxylase

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