Mutation Arg336 to Lys in <i>Saccharomyces cerevisiae</i> phosphoenolpyruvate carboxykinase originates an enzyme with increased oxaloacetate decarboxylase activity

Llanos, Liliana; Briones, René; Yévenes, Alejandro; González-Nilo, Fernando D.; Frey, Perry A.; Cardemil, Emilio

doi:10.1016/s0014-5793(01)02158-5

Cited by 22 publications

(8 citation statements)

References 18 publications

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“…The data obtained for the two secondary activities of mutant PEP carboxykinases indicate that the mutations at Lys213 or His233 elicit a modest affect on V max , in contrast to the large effects on V max for the primary reaction of the enzyme. These results agree with previous observations of this enzyme (9) and support the hypothesis that there are different ratelimiting steps for the primary reaction and the two secondary activities of PEP carboxykinase and the two "partial reactions" are not independent of each other. Mn 2+ Binding.…”

Section: Resultssupporting

confidence: 92%

“…Mutation of Lys 213 or His 233 in S. cereVisiae PEP carboxykinase caused only minor effects in V max of the pyruvate kinase-like and OAA decarboxylase activities of the enzyme, as compared with the effects in V max for the primary reaction. These observations are similar to previous findings reported with Arg 336 mutants of this same enzyme and suggest the existence of different rate-limiting steps for the primary reaction and for the secondary activities of S. cereVisiae PEP carboxykinase (9). Hence, the mutation of Lys 213 , His 233 , or Arg 336 of S. cereVisiae PEP carboxykinase appears to affect the rate of formation and the stabilization of enolpyruvate, the putative reaction intermediate of the PEP carboxykinase reaction (Scheme 1), thus affecting V max of the main reaction.…”

Section: Discussionsupporting

confidence: 92%

“…It is assumed that the pyruvate kinase-like and the OAA decarboxylase reactions reflect the phosphoryl transfer and decarboxylation steps of the main reaction, respectively, linked through the formation of enolpyruvate, a putative reaction intermediate (4,8), as depicted in Scheme 1. Catalysis of exchange of the methyl protons of pyruvate into solvent by S. cereVisiae PEP carboxykinase R336K supports the involvement of enolpyruvate as a reaction intermediate (9). Evidence for an essential role of Lys 256 of S. cereVisiae PEP carboxykinase in the phosphoryl transfer step has been provided (10).…”

mentioning

confidence: 82%

“…Site-Directed Mutagenesis and Enzyme Purification. Specific substitutions were introduced at the histidine and lysine triplets 233 and 213, respectively, in the cloned S. cereVisiae PEP carboxykinase gene (pMV7 plasmid) by the method of Ku ¨nkel (18) for His233Gln or by using recombinant PCR for Lys213Arg (9). To obtain the His233Gln substitution, synthetic oligonucleotide 5′-GCGGAAGAGTTCAAAGTTA-3′ (base substitution is shown in bold and underlined) was used, and all procedures were as previously described (10,18).…”

Section: Methodsmentioning

confidence: 99%

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Lysine 213 and Histidine 233 Participate in Mn(II) Binding and Catalysis in Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase

et al. 2002

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Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyses the reversible metal-dependent formation of oxaloacetate and ATP from PEP, ADP, and CO2 and plays a key role in gluconeogenesis. This enzyme also has oxaloacetate decarboxylase and pyruvate kinase-like activities. Mutations of PEP carboxykinase have been constructed where the residues Lys213 and His233, two residues of the putative Mn2+ binding site of the enzyme, were altered. Replacement of these residues by Arg and by Gln, respectively, generated enzymes with 1.9 and 2.8 kcal/mol lower Mn2+ binding affinity. Lower PEP binding affinity was inferred for the mutated enzymes from the protection effect of PEP against urea denaturation. Kinetic studies of the altered enzymes show at least a 5000-fold reduction in V(max) for the primary reaction relative to that for the wild-type enzyme. V(max) values for the oxaloacetate decarboxylase and pyruvate kinase-like activities of PEP carboxykinase were affected to a much lesser extent in the mutated enzymes. The mutated enzymes show a decreased steady-state affinity for Mn2+ and PEP. The results are consistent with Lys213 and His233 being at the Mn2+ binding site of S. cerevisiae PEP carboxykinase and the Mn2+ affecting the PEP interaction. The different effects of mutations in V(max) for the main reaction and the secondary activities suggest different rate-limiting steps for these reactions.

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Section: Resultssupporting

confidence: 92%

Section: Discussionsupporting

confidence: 92%

mentioning

confidence: 82%

Section: Methodsmentioning

confidence: 99%

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Lysine 213 and Histidine 233 Participate in Mn(II) Binding and Catalysis in Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase

et al. 2002

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“…Binding experiments were also carried out with oxalate, an analogue of enolpyruvate, the proposed reaction intermediate for PEP carboxykinases [20,23,24]. The incubation of the labeled enzyme with oxalate in the presence of Mn 2+ increased the fluorescence intensity by 50%, and the emission maximum was shifted to 386 nm.…”

Section: Effect Of Ligands On the P‐pyridoxyl Fluorescencementioning

confidence: 99%

Ligand interactions and protein conformational changes of phosphopyridoxyl‐labeled Escherichia coli phosphoenolpyruvate carboxykinase determined by fluorescence spectroscopy

Encinas

González-Nilo

Goldie

et al. 2002

European Journal of Biochemistry

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Escherichia coli phosphoenolpyruvate (PEP) carboxykinase catalyzes the decarboxylation of oxaloacetate and transfer of the c-phosphoryl group of ATP to yield PEP, ADP, and CO 2 . The interaction of the enzyme with the substrates originates important domain movements in the protein. In this work, the interaction of several substrates and ligands with E. coli PEP carboxykinase has been studied in the phosphopyridoxyl (P-pyridoxyl)-enzyme adduct. The derivatized enzyme retained the substrate-binding characteristics of the native protein, allowing the determination of several protein-ligand dissociation constants, as well as the role of Mg 2+ and Mn 2+ in substrate binding. The binding affinity of PEP to the enzyme-Mn 2+ complex was )8.9 kcalAEmol )1 , which is 3.2 kcalAEmol )1 more favorable than in the complex with Mg 2+ . For the substrate nucleotide-metal complexes, similar binding affinities ()6.0 to )6.2 kcalAEmol )1 ) were found for either metal ion. The fluorescence decay of the P-pyridoxyl group fitted to two lifetimes of 5.15 ns (34%) and 1.2 ns. These lifetimes were markedly altered in the derivatized enzyme-PEP-Mn complexes, and smaller changes were obtained in the presence of other substrates. Molecular models of the P-pyridoxyl-E. coli PEP carboxykinase showed different degrees of solvent-exposed surfaces for the P-pyridoxyl group in the open (substrate-free) and closed (substrate-bound) forms, which are consistent with acrylamide quenching experiments, and suggest that the fluorescence changes reflect the domain movements of the protein in solution.Keywords: Escherichia coli phosphoenolpyruvate carboxykinase; ligand binding; conformational changes; P-pyridoxyl fluorescence spectroscopy.Escherichia coli phosphoenolpyruvate carboxykinase [PEP carboxykinase; ATP:oxaloacetate carboxylase (trans-phosphorylating) EC 4.1.1.49] catalyzes the reversible decarboxylation of oxaloacetic acid (OAA) with the associated transfer of the c-phosphoryl group of ATP to yield PEP and ADP, where M 2+ is a divalent metal ion:The physiological role of this enzyme in bacteria and most other organisms is to catalyze the formation of PEP in the first committed step of gluconeogenesis [1]. The crystal structure of free-and substrate-bound E. coli PEP carboxykinase has been solved at 1.9 Å resolution [2,3]. The enzyme is a monomeric, globular protein that belongs to the a/b protein class. The overall structure has two domains, a 275 residue N-terminal domain, and a more compact 265 residue C-terminal domain, with the active site in a deep cleft between them. The recently reported crystal structure of Trypanosoma cruzi PEP-carboxykinase [4] shows remarkable similarity. Upon substrate binding, the E. coli enzyme undergoes a domain closure through a 20°rotation of the two domains towards each other, excluding bulk solvent from the active site and positioning active site residues for catalysis [3]. Results obtained with AlF 3 complexes of E. coli PEP carboxykinase indicate that phosphoryl transfer occurs via a direct displacement mec...

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In order to keep subscribers up‐to‐date with the latest developments in their field, this current awareness service is provided by John Wiley & Sons and contains newly‐published material on yeasts. Each bibliography is divided into 10 sections. 1 Books, Reviews & Symposia; 2 General; 3 Biochemistry; 4 Biotechnology; 5 Cell Biology; 6 Gene Expression; 7 Genetics; 8 Physiology; 9 Medical Mycology; 10 Recombinant DNA Technology. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted. (5 weeks journals ‐ search completed 9th May 2001)

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Mutation Arg336 to Lys in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase originates an enzyme with increased oxaloacetate decarboxylase activity

Cited by 22 publications

References 18 publications

Lysine 213 and Histidine 233 Participate in Mn(II) Binding and Catalysis in Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase

Lysine 213 and Histidine 233 Participate in Mn(II) Binding and Catalysis in Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase

Ligand interactions and protein conformational changes of phosphopyridoxyl‐labeled Escherichia coli phosphoenolpyruvate carboxykinase determined by fluorescence spectroscopy

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