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
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
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
Calcium-activated phosphoenolpyruvate carboxykinase from Escherichia coli is not inactivated by a number of sulfhydryl-directed reagents [5,5'-dithiobis(2-nitrobenzoate), iodoacetate, N-ethylmaleimide, N-(1-pyrenyl)maleimide or N-(iodoacetyl)-N'-(5-sulfo-1-naphthylethylenediamine)], unlike phosphoenolpyruvate carboxykinase from other organisms. On the other hand, the enzyme is rapidly inactivated by the arginyl-directed reagents 2,3-butanedione and 1-pyrenylglyoxal. The substrates, ADP plus PEP in the presence of Mn2+, protect the enzyme against inactivation by the diones. Quantitation of pyrenylglyoxal incorporation indicates that complete inactivation correlates with the binding of one inactivator molecule per mole of enzyme. Chemical modification by pyridoxal 5'-phosphate also produces inactivation of the enzyme, and the labeled protein shows a difference spectrum with a peak at 325 nm, characteristic of a pyridoxyl derivative of lysine. The inactivation by this reagent is also prevented by the substrates. Binding stoichiometries of 1.25 and 0.30 mol of reagent incorporated per mole of enzyme were found in the absence and presence of substrates, respectively. The results suggest the presence of functional arginyl and lysyl residues in or near the active site of the enzyme, and indicate lack of reactive functional sulfhydryl groups.
Assuntos
Arginina , Cisteína , Escherichia coli/enzimologia , Lisina , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Sítios de Ligação , Diacetil/farmacologia , Glioxal/análogos & derivados , Glioxal/farmacologia , Cinética , Fosfoenolpiruvato Carboxiquinase (GTP)/antagonistas & inibidores , Fosfoenolpiruvato Carboxiquinase (GTP)/química , Pirenos/farmacologia , Fosfato de Piridoxal/metabolismo , Fosfato de Piridoxal/farmacologia , Reagentes de Sulfidrila/farmacologiaRESUMO
Chicken liver mevalonate 5-diphosphate decarboxylase (ATP:(R)-5-diphosphomevalonate carboxy-lyase (dehydrating), EC 4.1.1.33) is inactivated by methylmethanethiosulfonate and 5,5'-dithiobis(2-nitrobenzoate). The presence of the substrates ATP or mevalonate 5-diphosphate protect very effectively against inactivation. The inactivation is second order with respect to methylmethanethiosulfonate, with an inactivation rate constant of (7.6 +/- 0.1).10(-5) microM-2.s-1, implying that the modifier may be reacting with more than one thiol in the enzyme. The enzyme is also inactivated by a number of dithiol-specific reagents, suggesting the presence of a functional dithiol. The determined pKapp values for enzyme modification by methyl methanethiosulfonate and phenylarsine oxide are 7.3 +/- 0.1 and 7.6 +/- 0.3, respectively. From the data presented, it is concluded that the enzyme possesses a functional dithiol that is important for substrate binding.
Assuntos
Carboxiliases/antagonistas & inibidores , Fígado/enzimologia , Reagentes de Sulfidrila/farmacologia , Tolueno/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Arsenicais/farmacologia , Galinhas , Ácido Ditionitrobenzoico/farmacologia , Ditiotreitol/farmacologia , Ativação Enzimática/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Cinética , Metanossulfonato de Metila/análogos & derivados , Metanossulfonato de Metila/farmacologia , Ácido Mevalônico/análogos & derivados , Ácido Mevalônico/farmacologiaRESUMO
The diastereoisomers of ATP alpha S and of ATP beta S have been used as substrate analogues for avian liver mevalonate 5-diphosphate decarboxylase. When the diastereoisomers of ATP alpha S were used, no reversal of the stereospecificity was seen upon changing Mg2+ for Cd2+, thus suggesting that the metal ion does not coordinate through the alpha-phosphoryl group of the nucleotide. Reversal of the stereospecificity, however, was observed when using the diastereoisomers of ATP beta S and upon changing Mg2+ by Zn2+ as the activating metal ion. Similar competitive inhibition constants for the diastereoisomers of MgATP beta S against MgATP were found. It is proposed that the active metal-nucleotide complex in catalysis is the lambda, beta-gamma MgATP complex.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Carboxiliases/metabolismo , Fígado/enzimologia , Trifosfato de Adenosina/química , Animais , Galinhas , Cinética , Conformação Molecular , Especificidade por SubstratoRESUMO
1. This work reviews the present knowledge of the physiological role and mechanism of action of mevalonate 5-diphosphate decarboxylase, the third enzyme involved in the biosynthesis of cholesterol from mevalonic acid. 2. Published evidence indicates that this and other enzymes of the cholesterol biosynthetic pathway present coordinate fluctuations in activity in rat liver. A possible regulatory role for the brain decarboxylases from chicken and rat has been proposed. 3. From kinetic and stereochemical studies with the chicken liver enzyme it has been proposed that the reaction is initiated by the abstraction of a proton from the 3-hydroxyl group of mevalonate 5-diphosphate by a basic group in the enzyme, followed by the nucleophilic attack of the C-3 oxygen on P gamma of the lambda isomer of the beta, gamma bidentate MgATP2- in a SN2(P) reaction that goes with inversion of configuration at P.