RESUMO
Anaerobiospirillum succiniciproducens phosphoenolpyruvate (PEP) carboxykinase catalyses the reversible metal-dependent formation of oxaloacetate (OAA) and ATP from PEP, ADP and CO(2). Mutations of PEP carboxykinase have been constructed where the residues His(225) and Asp(263), two residues of the enzyme's putative Mn(2+) binding site, were altered. Kinetic studies of the His225Glu, and Asp263Glu PEP carboxykinases show 600- and 16,800-fold reductions in V(max) relative to the wild-type enzyme, respectively, with minor alterations in K(m) for Mn(2+). Molecular modeling of wild-type and mutant enzymes suggests that the lower catalytic efficiency of the Asp263Glu enzyme could be explained by a movement of the lateral chain of Lys(248), a critical catalytic residue, away from the reaction center. The effect on catalysis of introducing a negatively charged oxygen atom in place of N(epsilon-2) at position 225 is discussed in terms of altered binding energy of the intermediate enolpyruvate.
Assuntos
Anaerobiospirillum/enzimologia , Manganês/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Substituição de Aminoácidos/genética , Anaerobiospirillum/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Ligação Proteica , Especificidade por Substrato/genéticaRESUMO
Comparative studies of intrinsic and extrinsic fluorescence of apyrases purified from two potato tuber varieties (Pimpernel and Desirée) were performed to determine differences in the microenvironment of the nucleotide binding site. The dissociation constants (K(d)) of Pimpernel apyrase for the binding of different fluorescent substrate analogs: methylanthranoyl (MANT-), trinitrophenyl (TNP-), and epsilon -derivatives of ATP and ADP were determined from the quenching of Trp fluorescence, and compared with K(d) values previously reported for Desirée enzyme. Binding of non-fluorescent substrate analogues decreased the Trp emission of both isoapyrases, indicating conformational changes in the vicinity of these residues. Similar effect was observed with fluorescent derivatives where, in the quenching effect, the transfer of energy from tryptophan residues to the fluorophore moiety could be additionally involved. The existence of energy transfer between Trp residues in the Pimpernel enzyme was demonstrated with epsilon -analogues, similar to our previous observations with the Desirée. From these results we deduced that tryptophan residues are close to or in the nucleotide binding site in both enzymes. Experiments with quenchers like acrylamide, Cs(+) and I(-), both in the presence and absence of nucleotide analogues, suggest the existence of differences in the nucleotide binding site of the two enzymes. From the results obtained in this work, we can conclude that the differences found in the microenvironment of the nucleotide binding site can explain, at least in part, the kinetic behaviour of both isoenzymes.
Assuntos
Apirase/metabolismo , Nucleotídeos/metabolismo , Solanum tuberosum/enzimologia , Triptofano/química , Acrilamida/química , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Apirase/química , Sítios de Ligação , Césio/química , Iodetos/química , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Nucleotídeos/química , Fotodegradação , Solanum tuberosum/química , Espectrometria de Fluorescência , Especificidade por SubstratoRESUMO
Plasmid pTbp60B (Kueng et al., J. Biol. Chem. 264 (1989) 5203-5209) was employed to obtain, through the polymerase chain reaction, the Trypanosoma brucei gene coding for phosphoenolpyruvate (PEP) carboxykinase, and then cloned into the yeast expression plasmid pYES2. The cloned gene was completely sequenced and the expression plasmid transformed into Saccharomyces cerevisiae PUK-3B (MATalpha pck1 ura3 ade1) competent cells. Gene expression took place upon induction with 2% galactose, and the recombinant T. brucei PEP carboxykinase was purified to near homogeneity. The basic molecular and catalytic characteristics of the recombinant enzyme were determined, and they showed to be essentially similar to those reported for wild type T. brucei PEP carboxykinase (Hunt and Köhler, Biochim. Biophys. Acta 1249 (1995) 15-22). The expression system here described is a reliable non-pathogenic source of T. brucei PEP carboxykinase.
Assuntos
Vetores Genéticos , Fosfoenolpiruvato Carboxiquinase (ATP)/biossíntese , Saccharomyces cerevisiae/genética , Trypanosoma brucei brucei/enzimologia , Animais , Clonagem Molecular/métodos , Expressão Gênica , Genes de Protozoários , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/isolamento & purificação , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Reação em Cadeia da Polimerase/métodos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Trypanosoma brucei brucei/genéticaRESUMO
Lysine 256, a conserved amino acid of Saccharomycescerevisiae phosphoenolpyruvate (PEP) carboxykinase located in the consensus kinase 1a sequence of the enzyme, was changed to alanine, arginine, or glutamine by site-directed mutagenesis. These substitutions did not result in gross changes in the protein structure, as indicated by circular dichroism, tryptophan fluorescence spectroscopy, and gel-exclusion chromatography. The three variant enzymes showed almost unaltered Km for MnADP but about a 20 000-fold decrease in Vmax for the PEP carboxylation reaction, as compared to wild-type PEP carboxykinase. The variant enzymes presented oxaloacetate decarboxylase activity at levels similar to those of the native protein; however, they lacked pyruvate kinase-like activity. The dissociation constant for the enzyme-MnATP complex was 1.3 +/- 0.3 microM for wild-type S. cerevisiae PEP carboxykinase, and the corresponding values for the Lys256Arg, Lys256Gln, and Lys256Ala mutants were 2.0 +/- 0.6 microM, 17 +/- 2 microM, and 20 +/- 6 microM, respectively. These results collectively show that a positively charged residue is required for proper binding of MnATP and that Lys256 plays an essential role in transition state stabilization during phosphoryl transfer for S. cerevisiae PEP carboxykinase.
Assuntos
Lisina/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Saccharomyces cerevisiae/enzimologia , Trifosfato de Adenosina/metabolismo , Dicroísmo Circular , Cristalografia por Raios X , Cinética , Lisina/análise , Modelos Moleculares , Fosfoenolpiruvato Carboxiquinase (ATP)/químicaRESUMO
Escherichia coli and Saccharomyces cerevisiae phospho enol pyruvate (PEP) carboxykinases are inactivated by diethylpyrocarbonate (DEP). Inactivation follows pseudo-first-order kinetics and exhibits a second order rate constant of 0.8 M-1 s-1 for the bacterial enzyme and of 3.3 M-1 s-1 for the yeast carboxykinase. A mixture of ADP + PEP + MnCl2 protects against inactivation by DEP, suggesting that residues within the active site are being modified. After digestion of the modified proteins with trypsin, the labeled peptides were isolated by reverse-phase high-performance liquid chromatography and sequenced by Edman degradation. His-271 of E. coli carboxykinase and His-273 of the yeast enzyme were identified as the reactive amino-acid residues. The modified histidine residues occupy equivalent positions in these enzymes, and they are located in a highly conserved region of all ATP-dependent phospho enol pyruvate carboxykinases described so far.
Assuntos
Escherichia coli/enzimologia , Fosfoenolpiruvato Carboxiquinase (GTP)/química , Saccharomyces cerevisiae/enzimologia , Difosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , Dietil Pirocarbonato/farmacologia , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Histidina/química , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fosfoenolpiruvato/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/antagonistas & inibidores , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Especificidade por SubstratoRESUMO
The reaction of Woordward's reagent K (WRK) with model amino acids and proteins has been analyzed. Our results indicate that WRK forms 340-nm-absorbing adducts with sulfhydryl- and imidazol-containing compounds, but not with carboxylic acid derivatives, in agreement with Liamas et al. [(1986), J. Am. Chem. Soc. 108, 5543-5548], but not with Sinha and Brewer [(1985), Anal. Biochem. 151, 327-333]. The chemical modification of Escherichia coli and Saccharomyces cerevisiae phosphoenolpyruvate carboxykinases with WRK leads to an increase in the absorption at 340 nm, and we have demonstrated its reaction with His and Cys residues in these proteins. These results caution against claims of glutamic or aspartic acid modification by WRK based on the absorption at 340 nm of protein- WRK adducts.
Assuntos
Cisteína/química , Histidina/química , Indicadores e Reagentes/química , Isoxazóis/química , Fosfoenolpiruvato Carboxiquinase (GTP)/química , Aminoácidos/análise , Sítios de Ligação , Escherichia coli/enzimologia , Cinética , Saccharomyces cerevisiae/enzimologia , Espectrofotometria UltravioletaRESUMO
Important information on enzyme ligand interactions can be obtained when analyzing the kinetics of chemical modification reactions. In this article, several kinetic models of inactivation are discussed, along with the determination of enzyme-ligand dissociation constants and of the pK of enzyme reactive groups from chemical modification kinetic data.
Assuntos
Ativação Enzimática/fisiologia , Enzimas/metabolismo , CinéticaRESUMO
Important information on enzyme ligand interactions can be obtained when analyzing the kinetics of chemical modification reactions. In this article, several kinetic models of inactivation are discussed, along with the determination of enzyme-ligand dissociation constants and of the pK of enzyme reactive groups from chemical modification kinetic data
Assuntos
Ativação Enzimática/fisiologia , Enzimas/metabolismo , CinéticaRESUMO
Escherichia coli and Saccharomyces cerevisiae phosphoenolpyruvate carboxykinases (PEPCKs), were inactivated by pyridoxal 5'-phosphate followed by reduction with sodium borohydride. Concomitantly with the inactivation, one pyridoxyl group was incorporated in each enzyme monomer. The modification and loss of activity was prevented in the presence of ADP plus Mn2+. After digestion of the modified protein with trypsin plus protease V-8, the labeled peptides were isolated by reverse-phase high-performance liquid chromatography and sequenced by gas-phase automatic Edman degradation. Lys286 of bacterial PEPCK and Lys289 of the yeast enzyme were identified as the reactive amino acid residues. The modified lysine residues are conserved in all ATP-dependent phosphoenolpyruvate carboxykinases described so far.
Assuntos
Fosfoenolpiruvato Carboxiquinase (GTP)/química , Sequência de Aminoácidos , Sítios de Ligação , Escherichia coli/enzimologia , Lisina/química , Dados de Sequência Molecular , Peptídeos/química , Fosfoenolpiruvato Carboxiquinase (GTP)/antagonistas & inibidores , Fosfato de Piridoxal/química , Saccharomyces cerevisiae/enzimologia , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Each of the four subunits of the Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase has one cysteine residue (Cys-364) that is protected against alkylation by MnATP and that is thought to be located at (or close to) the active site (M. Alvear, M. V. Encinas, S. Latshaw, R. G. Kemp, and E. Cardemil, 1992, Biochim. Biophys. Acta 1119, 35-38). To determine the distance relationships between these residues within this tetrameric enzyme, we have derivatized one of these reactive thiols with N-acetyl-N'-(5-sulfo-1-naphthyl) ethylenediamine (AEDANS) and the others progressively with 4-[N-[(acetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole (ANBD). In the doubly labeled protein nonradiative singlet-singlet energy transfer between AEDANS (donor) and ANBD (acceptor) was observed. The efficiency of the energy transfer is proportional to the number of occupied acceptor sites. From these data it has been determined that one of the acceptor sites is 33 A from the donor, and the remaining two sites are 44-46 A from the donor. Cross-linking experiments revealed that mainly cross-linked dimers were produced upon reaction of the enzyme with o-phthalaldehyde and dithiobissuccinimidylpropionate. We interpret these results as an indication that this tetrameric enzyme is most likely composed of an association of two dimers.