RESUMEN
Residues 1--10 of porcine fructose-1,6-bisphosphatase (FBPase) are poorly ordered or are in different conformations, sensitive to the state of ligation of the enzyme. Deletion of the first 10 residues of FBPase reduces k(cat) by 30-fold and Mg(2+) affinity by 20-fold and eliminates cooperativity in Mg(2+) activation. Although a fluorescent analogue of AMP binds with high affinity to the truncated enzyme, AMP itself potently inhibits only 50% of the enzyme activity. Additional inhibition occurs only when the concentration of AMP exceeds 10 mm. Deletion of the first seven residues reduces k(cat) and Mg(2+) affinity significantly but has no effect on AMP inhibition. The mutation of Asp(9) to alanine reproduces the weakened affinity for Mg(2+) observed in the deletion mutants, and the mutation of Ile(10) to aspartate reproduces the AMP inhibition of the 10-residue deletion mutant. Changes in the relative stability of the known conformational states for loop 52--72, in response to changes in the quaternary structure of FBPase, can account for the phenomena above. Some aspects of the proposed model may be relevant to all forms of FBPase, including the thioredoxin-regulated FBPase from the chloroplast.
Asunto(s)
Fructosa-Bifosfatasa/química , Regulación Alostérica , Animales , Catálisis , Fluorescencia , Fructosa-Bifosfatasa/antagonistas & inhibidores , Fructosa-Bifosfatasa/metabolismo , Fructosadifosfatos/farmacología , Magnesio/farmacología , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Relación Estructura-Actividad , PorcinosAsunto(s)
Proteínas de Escherichia coli , Escherichia coli/enzimología , Expresión Génica , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/aislamiento & purificación , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Clonación Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Brain hexokinase (HKI) is inhibited potently by its product glucose 6-phosphate (G6P); however, the mechanism of inhibition is unsettled. Two hypotheses have been proposed to account for product inhibition of HKI. In one, G6P binds to the active site (the C-terminal half of HKI) and competes directly with ATP, whereas in the alternative suggestion the inhibitor binds to an allosteric site (the N-terminal half of HKI), which indirectly displaces ATP from the active site. Single mutations within G6P binding pockets, as defined by crystal structures, at either the N- or C-terminal half of HKI have no significant effect on G6P inhibition. On the other hand, the corresponding mutations eliminate product inhibition in a truncated form of HKI, consisting only of the C-terminal half of the enzyme. Only through combined mutations at the active and allosteric sites, using residues for which single mutations had little effect, was product inhibition eliminated in HKI. Evidently, potent inhibition of HKI by G6P can occur from both active and allosteric binding sites. Furthermore, kinetic data reported here, in conjunction with published equilibrium binding data, are consistent with inhibitory sites of comparable affinity linked by a mechanism of negative cooperativity.
Asunto(s)
Sitio Alostérico/efectos de los fármacos , Encéfalo/enzimología , Dominio Catalítico/efectos de los fármacos , Glucosa-6-Fosfato/farmacología , Hexoquinasa/antagonistas & inhibidores , Hexoquinasa/genética , Humanos , Cinética , Modelos Químicos , Modelos Moleculares , Mutagénesis Sitio-DirigidaRESUMEN
Asn64, Asp68, Lys71, Lys72, and Asp74 of porcine liver fructose-1, 6-bisphosphatase (FBPase) are conserved residues and part of a loop for which no electron density has been observed in crystal structures. Yet mutations of the above dramatically affect catalytic rates and/or AMP inhibition. The Asp74 --> Ala and Asp74 --> Asn mutant enzymes exhibited 50,000- and 2,000-fold reductions, respectively, in kcat relative to wild-type FBPase. The pH optimum for the catalytic activity of the Asp74 --> Glu, Asp68 --> Glu, Asn64 --> Gln, and Asn64 --> Ala mutant enzymes shifted from pH 7.0 (wild-type enzyme) to pH 8.5, whereas the Lys71 --> Ala mutant and Lys71,72 --> Met double mutant had optimum activity at pH 7.5. Mg2+ cooperativity, Km for fructose 1,6-bisphosphate, and Ki for fructose 2,6-bisphosphate were comparable for the mutant and wild-type enzymes. Nevertheless, for the Asp74 --> Glu, Asp68 --> Glu, Asn64 --> Gln, and Asn64 --> Ala mutants, the binding affinity for Mg2+ decreased by 40-125-fold relative to the wild-type enzyme. In addition, the Asp74 --> Glu and Asn64 --> Ala mutants exhibited no AMP cooperativity, and the kinetic mechanism of AMP inhibition with respect to Mg2+ was changed from competitive to noncompetitive. The double mutation Lys71,72 --> Met increased Ki for AMP by 175-fold and increased Mg2+ affinity by 2-fold relative to wild-type FBPase. The results reported here strongly suggest that loop 51-72 is important for catalytic activity and the mechanism of allosteric inhibition of FBPase by AMP.
Asunto(s)
Adenosina Monofosfato/farmacología , Fructosa-Bifosfatasa/metabolismo , Mutagénesis Sitio-Dirigida , Regulación Alostérica , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Catálisis , Cartilla de ADN , Fructosa-Bifosfatasa/antagonistas & inhibidores , Cinética , PorcinosAsunto(s)
Endorribonucleasas/química , Ribonucleasas/química , Proteínas Bacterianas , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Endorribonucleasas/genética , Concentración de Iones de Hidrógeno , Conformación Proteica , Desnaturalización Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Ribonucleasas/genética , TermodinámicaAsunto(s)
Bacillus/enzimología , Endorribonucleasas/metabolismo , Ribonucleasas/metabolismo , Fusión Artificial Génica , Proteínas Bacterianas , ADN Bacteriano/genética , ADN Recombinante/análisis , ADN Recombinante/genética , Endorribonucleasas/genética , Cinética , Mutagénesis , Ribonucleasas/genética , Especificidad por SustratoAsunto(s)
Endorribonucleasas/genética , Hormona del Crecimiento/genética , Proteínas Recombinantes de Fusión/genética , Ribonucleasas/genética , Animales , Proteínas Bacterianas , Secuencia de Bases , ADN , Cartilla de ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Datos de Secuencia Molecular , Mutagénesis , Reacción en Cadena de la Polimerasa , PorcinosRESUMEN
We have reported previously about the cloning of the binase gene in E. coli. In this work, using an original approach named "homolog gene recombination" method (HGR), vectors for binase expression in E. coli have been constructed. Transcription of the binase gene have been directed through either tac-promoter or PR-promoter of bacteriophage lambda under the control of temperature-sensitive CI857 repressor. The last promoter gave the maximum yield of binase, up to 100 mg of protein per litre of heat-induced bacterial culture. The location of the transcription terminator at the 3' terminus of the binase gene raised the expression approximately two times more. A chromatographic method have been developed and applied for the control of binase accumulation in growth medium without measuring the ribonuclease activity.