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1.
Eur J Biochem ; 268(12): 3558-65, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11422387

RESUMO

Pyruvate decarboxylase (PDC, EC 4.1.1.1) is a thiamin diphosphate-dependent enzyme about which there is a large body of structural and functional information. The active site contains several absolutely conserved ionizable groups and all of these appear to be important, as judged by the fact that mutation diminishes or abolishes catalytic activity. Previously we have shown [Schenk, G., Leeper, F.J., England, R., Nixon, P.F. & Duggleby, R.G. (1997) Eur. J. Biochem. 248, 63-71] that the activity is pH-dependent due to changes in kcat/Km while kcat itself is unaffected by pH. The effect on kcat/Km is determined by a group with a pKa of 6.45; the identity of this group has not been determined, although H113 is a possible candidate. Here we mutate five crucial residues in the active site with ionizable side-chains (D27, E50, H113, H114 and E473) in turn, to residues that are nonionizable or should have a substantially altered pKa. Each protein was purified and characterized kinetically. Unexpectedly, the pH-dependence of kcat/Km is largely unaffected in all mutants, ruling out the possibility that any of these five residues is responsible for the observed pKa of 6.45. We conjecture that the kcat/Km profile reflects the protonation of an alcoholate anion intermediate of the catalytic cycle.


Assuntos
Piruvato Descarboxilase/metabolismo , Zymomonas/enzimologia , Sítios de Ligação , Concentração de Íons de Hidrogênio , Cinética , Mutagênese Sítio-Dirigida , Piruvato Descarboxilase/genética , Piruvato Descarboxilase/isolamento & purificação
2.
Mol Genet Metab ; 72(3): 269-72, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11243735

RESUMO

One cause of congenital lactic acidosis is a mutation in the E1 alpha-subunit of the pyruvate dehydrogenase multienzyme complex. Little is known about the consequences of these mutations at the enzymatic level. Here we study the A199T mutation by expressing the protein in Escherichia coli. The specific activity is 25% of normal and the K(m) for pyruvate is elevated by 10-fold. Inhibitors of lactate dehydrogenase might be a useful therapy for patients with such mutations.


Assuntos
Acidose Láctica , Acidose Láctica/congênito , Mutação , Piruvato Desidrogenase (Lipoamida) , Complexo Piruvato Desidrogenase , Complexo Piruvato Desidrogenase/genética , Acidose Láctica/genética , Escherichia coli/genética , Humanos , Mutagênese , Complexo Piruvato Desidrogenase/metabolismo , Transfecção
3.
Brain Res ; 892(1): 218-27, 2001 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-11172768

RESUMO

Glucose loading of rats made thiamin deficient by dietary deprivation of thiamin and the administration of pyrithiamin (40 microg/100 g, i.p.) precipitates an acute neuropathy, a model of Wernicke's encephalopathy in man (Zimitat and Nixon, Metab. Brain Dis. 1999;14:1-20). Immunohistochemical detection of Fos proteins was used as a marker to identify neuronal populations in the thiamin-deficient rat brain affected by glucose loading. As thiamin deficiency progressed, the extent and intensity of Fos-like immunoreactivity (FLI) in brain structures typically affected by thiamin deficiency (the thalamus, mammillary bodies, inferior colliculus, vestibular nucleus and inferior olives) were markedly increased when compared to thiamin-replete controls. Glucose loading for 1-3 days further increased the intensity of FLI in these same regions, consistent with a dependence of Fos expression on carbohydrate metabolism as well as on thiamin deficiency. The timed acute changes that follow a bolus glucose load administered to thiamin-deficient animals may provide a sequential account of events in the pathogenesis of brain damage in this model of Wernicke's encephalopathy.


Assuntos
Encéfalo/metabolismo , Regulação da Expressão Gênica/fisiologia , Genes Precoces , Genes fos , Deficiência de Tiamina/fisiopatologia , Encefalopatia de Wernicke/fisiopatologia , Animais , Antimetabólitos/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Glucose/farmacologia , Humanos , Especificidade de Órgãos , Piritiamina/farmacologia , Ratos , Ratos Wistar , Deficiência de Tiamina/genética , Deficiência de Tiamina/patologia , Encefalopatia de Wernicke/genética , Encefalopatia de Wernicke/patologia
4.
Eur J Biochem ; 267(21): 6493-500, 2000 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11029594

RESUMO

Pyruvate decarboxylase (PDC) is one of several enzymes that require thiamin diphosphate (ThDP) and a bivalent cation as essential cofactors. The three-dimensional structure of PDC from Zymomonas mobilis (ZMPDC) shows that Asp27 (D27) is close to ThDP in the active site, and mutagenesis of this residue has suggested that it participates in catalysis. The normal product of the PDC reaction is acetaldehyde but it is known that the enzyme can also form acetoin as a by-product from the hydroxyethyl-ThDP reaction intermediate. This study focuses on the role of D27 in the production of acetoin and a second by-product, acetolactate. D27 in ZMPDC was altered to alanine (D27A) and this mutated protein, the wild-type, and two other previously constructed PDC mutants (D27E and D27N) were expressed and purified. Determination of the kinetic properties of D27A showed that the affinity of D27A for ThDP is decreased 30-fold, while the affinity for Mg2+ and the Michaelis constant for pyruvate were similar to those of the wild-type. The time-courses of their reactions were investigated. Each mutant has greatly reduced ability to produce acetaldehyde and acetoin compared with the wild-type PDC. However, the effect of these mutations on acetaldehyde production is greater than that on acetoin formation. The D27A mutant can also form acetolactate, whereas neither of the other mutants, nor the wild-type PDC, can do so. In addition, acetaldehyde formation and/or release are reversible in wild-type ZMPDC but irreversible for the mutants. The results are explained by a mechanism involving thermodynamic and geometric characteristics of the intermediates in the reaction.


Assuntos
Acetoína/metabolismo , Substituição de Aminoácidos/genética , Ácido Aspártico/metabolismo , Lactatos/metabolismo , Piruvato Descarboxilase/metabolismo , Zymomonas/enzimologia , Acetaldeído/farmacologia , Ácido Aspártico/genética , Sítios de Ligação , Catálise/efeitos dos fármacos , Cinética , Modelos Químicos , Modelos Moleculares , Mutação/genética , Conformação Proteica , Piruvato Descarboxilase/química , Piruvato Descarboxilase/genética , Piruvato Descarboxilase/isolamento & purificação , Ácido Pirúvico/metabolismo , Ácido Pirúvico/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Zymomonas/genética
5.
Biochemistry ; 39(31): 9430-7, 2000 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-10924138

RESUMO

The three-dimensional structure of Zymomonas mobilis pyruvate decarboxylase shows that the carboxyl-terminal region of the protein occludes the active site. This observation is consistent with earlier suggestions that the active site is inaccessible to solvent during catalysis. However, the carboxyl-terminal region must move aside to allow entry of the substrate, and again to permit the products to leave. Here we have examined the role of the carboxyl terminus by making 15 variants of the enzyme with serial deletions. The activity is largely unaffected by removal of up to seven residues but deletion of the next two, R561 and S560, results in a drastic loss of activity. Five of these deletion mutants were purified and fully characterized and showed progressive decreases in activity, in the ability to discriminate between pyruvate and larger substrates, and in cofactor affinity. Several substitution mutants at residues R561 and S560 were prepared, purified, and fully characterized. The results indicate important roles for the side-chain of R561 and the backbone atoms of S560. It is suggested that the carboxyl-terminal region of pyruvate decarboxylase is needed to lock in the cofactors and for the proper closure of the active site that is required for discrimination between substrates and for decarboxylation to occur.


Assuntos
Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Piruvato Descarboxilase/genética , Piruvato Descarboxilase/metabolismo , Deleção de Sequência , Zymomonas/enzimologia , Zymomonas/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Arginina/genética , Sítios de Ligação/genética , Coenzimas/metabolismo , Ativação Enzimática/genética , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Serina/genética , Especificidade por Substrato/genética
6.
Metab Brain Dis ; 14(1): 1-20, 1999 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10348310

RESUMO

A rat model of glucose-precipitated Wernicke's encephalopathy (WE) has been developed in which glucose loading (10 g/kg, i.p.) of ataxic thiamin-deficient (TD) rats induced episodes of gross neurological dysfunction and sometimes death. The acute effects of a glucose load on the neurological state of thiamin-replete control and TD rats were assessed by scoring of clinical observations and performance measured on a moving belt (MB) apparatus at 30 min intervals for 2 hr after the challenge. Glucose loading or saline treatment (2.5 mL, i.p.) had no significant behavioural or clinical consequences when administered to controls or rats fed TD diet for <21 days. Glucose loading of ataxic rats fed TD diet for 28-35 days precipitated episodes of gross ataxia and signs of advanced neurological dysfunction (e.g. loss of righting reflex and hyperexcitability) leading to significant increases in the Ataxia (p<0.05) and Advanced Sign (p<0.05) scores within 2 hr after the challenge. Simultaneously, the performance of these animals on the MB decreased 10-fold. Regular glucose challenges significantly increased the rate of progression of disease in TD rats when compared with untreated TD rats. This model may be useful for the further investigation of the pathogenesis of WE at the molecular level.


Assuntos
Encefalopatias/induzido quimicamente , Encefalopatias/etiologia , Glucose , Deficiência de Tiamina/complicações , Doença Aguda , Animais , Progressão da Doença , Feminino , Glucose/farmacologia , Atividade Motora/fisiologia , Ratos , Ratos Wistar , Valores de Referência , Deficiência de Tiamina/fisiopatologia
7.
Biochem J ; 339 ( Pt 2): 255-60, 1999 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-10191255

RESUMO

Zymomonas mobilis pyruvate decarboxylase (EC 4.1.1.1) was subjected to site-directed mutagenesis at two acidic residues near the thiamin diphosphate cofactor in the active site. Asp-27 was changed to Glu or Asn, and Glu-473 was mutated to Asp (E473D) or Gln (E473Q). Each mutant protein was purified to near-homogeneity, and the kinetic and cofactor-binding properties were compared with those of the wild-type protein. Despite the very conservative nature of these alterations, all mutants had a very low, but measurable, specific activity ranging from 0.025% (E473Q) to 0.173% (E473D) of the wild type. With the exception of E473Q, the mutants showed small decreases in the affinity for thiamin diphosphate, and binding of the second cofactor (Mg2+) was also weakened somewhat. With E473Q, both cofactors seemed to be very tightly bound so that they were not removed by the treatment that was effective for the wild-type enzyme and other mutant forms. All mutants showed minor changes in the Km for substrate, but these alterations did not account for the low activities. These low specific activities, accompanied by little change in the Km for pyruvate, are consistent with a quantitative model of the catalytic cycle in which the main effect of the mutations is to slow the decarboxylation step with a minor change in the rate constant for pyruvate binding.


Assuntos
Ácido Aspártico/metabolismo , Ácido Glutâmico/metabolismo , Piruvato Descarboxilase/metabolismo , Zymomonas/enzimologia , Catálise , Eletroforese em Gel de Poliacrilamida , Cinética , Mutagênese Sítio-Dirigida , Conformação Proteica , Piruvato Descarboxilase/química , Piruvato Descarboxilase/genética , Especificidade por Substrato , Tiamina Pirofosfato/metabolismo
8.
FEBS Lett ; 437(3): 273-7, 1998 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-9824306

RESUMO

The pyruvate dehydrogenase complex catalyzes the conversion of pyruvate to acetyl-CoA. The first component (E1) converts pyruvate to bound acetaldehyde using thiamine diphosphate (ThDP) and Mg2+ as cofactors. There is no 3D structure of E1 available but those of other ThDP-dependent enzymes show some similarities including a glutamate residue that assists in ThDP activation. Eukaryotic E1 has an alpha2beta2 structure and the conserved Glu89 of the beta-subunit was identified as a possible catalytic residue by sequence alignment. Human E1 was expressed in Escherichia coli and purified. Mutating Glu89 to glutamine, aspartate and alanine markedly reduces catalytic activity and the affinity for ThDP, consistent with a role as the catalytic glutamate.


Assuntos
Domínio Catalítico/genética , Ácido Glutâmico/metabolismo , Complexo Piruvato Desidrogenase/metabolismo , Sequência de Aminoácidos , Humanos , Cinética , Dados de Sequência Molecular , Ligação Proteica , Piruvato Desidrogenase (Lipoamida) , Complexo Piruvato Desidrogenase/biossíntese , Complexo Piruvato Desidrogenase/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Tiamina Pirofosfato/metabolismo
9.
Brain Res ; 791(1-2): 347-51, 1998 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-9593988

RESUMO

Using T2-weighted Magnetic Resonance Imaging (MRI) in a pyrithiamin-treated, thiamin deficient (TD) rat model of Wernicke's encephalopathy (WE), we have observed hyperintensity in the thalamus, hypothalamus, collicular bodies and hippocampus which was enhanced 40 min after a glucose load. Hyperintensity was not evident in these structures in thiamin replete rats receiving glucose nor was it enhanced in TD rats administered 2-deoxyglucose. Residual hyperintensity was still evident in the hippocampus as long as 30 days after thiamin administration and was increased by repeat glucose challenge at that time. These data indicate that the hippocampus is as vulnerable as the thalamus to some persistent pathological change when glucose is metabolised in a state of thiamin deficiency.


Assuntos
Glucose/farmacologia , Hipocampo/efeitos dos fármacos , Imageamento por Ressonância Magnética , Deficiência de Tiamina/diagnóstico , Encefalopatia de Wernicke/diagnóstico , Análise de Variância , Animais , Modelos Animais de Doenças , Feminino , Hipocampo/patologia , Ratos , Ratos Wistar
10.
Int J Biochem Cell Biol ; 30(3): 369-78, 1998 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-9611778

RESUMO

Transketolase belongs to the family of thiamin diphosphate dependent enzymes. The aim of this study was to establish a bacterial expression system for human transketolase in order to investigate the functional characteristics of mammalian transketolases. The level of recombinant human enzyme expressed in Escherichia coli was modest. Purification of recombinant transketolase and separation from the E. coli enzyme has been greatly simplified by means of a non-cleavable hexa-histidine tag. The highest specific activity was 13.5 U/mg and the K(m) values were 0.27 +/- 0.02 and 0.51 +/- 0.05 mM for the substrates D-xylulose 5-phosphate and D-ribose 5-phosphate, respectively. Binding of cofactors to the apoenzyme showed the expected hysteresis. Without preincubation, the K(m) values for thiamin diphosphate and for Mg2+ were, respectively, 4.1 +/- 0.8 and 2.5 +/- 0.4 microM, but after 1 h of preincubation these values were 85 +/- 16 nM and 0.74 +/- 0.23 microM. The kinetic constants are similar to those of the native enzyme purified from human erythrocytes. Despite the modest expression level the reported system is well suited to a variety of functional studies.


Assuntos
Transcetolase/genética , Clonagem Molecular , DNA Complementar/genética , Escherichia coli/genética , Expressão Gênica , Humanos , Cinética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Transcetolase/isolamento & purificação , Transcetolase/metabolismo
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