Thermostability of yeast hexokinase and yeast glucose-6-phosphate dehydrogenase

Zaitzeva, E. A.; Chukrai, E. S.; Poltorak, O. M.

doi:10.1007/bf02785689

Cited by 10 publications

(7 citation statements)

References 5 publications

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“…In these cell extracts the hexose transporters, hexokinase (HXK), fructose-1,6-bisphosphate aldolase, and PGK did not show a significant change in V max when assayed at both temperatures. To show that we were not observing an artifact introduced by enzyme inactivation during the assay, we tested the activity of HXK, which belongs to the most thermolabile enzymes (37), after incubation of cell-free extracts from steady states at both cultivation temperatures at 38°C for 30 min. No significant decrease in enzyme capacity was observed (data not shown).…”

mentioning

confidence: 99%

Quantitative Analysis of the High Temperature-induced Glycolytic Flux Increase in Saccharomyces cerevisiae Reveals Dominant Metabolic Regulation

Postmus

Canelas²,

Bouwman

et al. 2008

Journal of Biological Chemistry

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A major challenge in systems biology lies in the integration of processes occurring at different levels, such as transcription, translation, and metabolism, to understand the functioning of a living cell in its environment. We studied the high temperatureinduced glycolytic flux increase in Saccharomyces cerevisiae and investigated the regulatory mechanisms underlying this increase. We used glucose-limited chemostat cultures to separate regulatory effects of temperature from effects on growth rate. Growth at increased temperature (38°C versus 30°C) resulted in a strongly increased glycolytic flux, accompanied by a switch from respiration to a partially fermentative metabolism. We observed an increased flux through all enzymes, ranging from 5-to 10-fold. We quantified the contributions of direct temperature effects on enzyme activities, the gene expression cascade and shifts in the metabolic network, to the increased flux through each enzyme. To do this we adapted flux regulation analysis. We show that the direct effect of temperature on enzyme kinetics can be included as a separate term. Together with hierarchical regulation and metabolic regulation, this term explains the total flux change between two steady states. Surprisingly, the effect of the cultivation temperature on enzyme catalytic capacity, both directly through the Arrhenius effect and indirectly through adapted gene expression, is only a moderate contribution to the increased glycolytic flux for most enzymes. The changes in flux are therefore largely caused by changes in the interaction of the enzymes with substrates, products, and effectors.Microorganisms encounter environmental changes, which they have to withstand and adapt to in order to survive. Changes in ambient temperature are common to almost every ecological niche. Temperature influences the structural and functional properties of cellular components, both physically and chemically. Physically, temperature affects membrane fluidity (1, 2) and diffusion rates, as well as protein folding and stability (3). Chemically, temperature directly affects reaction rates in the cell. This study focuses on the adaptation of cells to temperatures higher than that optimal for growth.Microbes adapt to high temperature by altering their cellular make-up such as lipid composition, membrane fluidity, and the induction of large numbers of heat shock genes (3-11), which have a wide variety of functions. Many encode protein chaperones involved in protein (un)folding (12, 13) or degradation of damaged proteins (14). Others are involved in the synthesis of the thermoprotecting disaccharide trehalose, which is known to be involved in stabilization of membranes and proteins (15, 16) as well as in storage of free energy (17). Many of these adaptive responses put a significant additional energy burden on the cells (18).There still is little clarity on the actual mechanisms by which cells maintain a balance between the energy needs for adaptive responses to stress survival and those for processes indispensable for grow...

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mentioning

confidence: 99%

Quantitative Analysis of the High Temperature-induced Glycolytic Flux Increase in Saccharomyces cerevisiae Reveals Dominant Metabolic Regulation

Postmus

Canelas²,

Bouwman

et al. 2008

Journal of Biological Chemistry

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“…Data analysis was performed according to the dissociative-thermal-inactivation theory (Zaitzeva et al, 1996;Poltorak et al, 1998). For dimeric enzymes, it can be explained according to the following scheme:…”

Section: Kinetics Of Thermal Inactivationmentioning

confidence: 99%

“…Because of some substantial structural alteration, E den is unable to be reassociated as E 2 . The k 1 , k -1 and k den values can be obtained by the plot ln(v/v 0 ) versus time under denaturing conditions (Zaitzeva et al, 1996). For obtaining the k values, a specific range of temperature (from 3 to 7 o C over T opt ) and a protein concentration around the numerical value of dissociation constant for dimeric protein are necessary.…”

Section: Kinetics Of Thermal Inactivationmentioning

confidence: 99%

“…The logarithm of the remaining activity percentage is primarily assumed to be a linear function of the time at any temperature, even if it is not linear (Sriprapundh et al, 2000). At particular ranges of temperature and enzyme concentration, the non-linear behavior of the thermal inactivation of oligomeric enzyme is imposed, and contains large structural information (Zaitzeva et al, 1996;Poltorak et al, 1998).…”

Section: Kinetics Of Lsao Thermal Inactivationmentioning

confidence: 99%

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Conformational Lock and Dissociative Thermal Inactivation of Lentil Seedling Amine Oxidase

Moosavi-Nejad¹,

Moosavi-Movahedi²,

Rezaei‐Tavirani³

et al. 2003

BMB Reports

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The kinetics of thermal inactivation of copper-containing amine oxidase from lentil seedlings were studied in a 100 mM potassium phosphate buffer, pH 7, using putrescine as the substrate. The temperature range was between 47-60 o C. The thermal inactivation curves were not linear at 52 and 57 o C; three linear phases were shown. The first phase gave some information about the number of dimeric forms of the enzyme that were induced by the higher temperatures using the "conformational lock" pertaining theory to oligomeric enzyme. The "conformational lock" caused two additional dimeric forms of the enzyme when the temperature increased to 57 o C. The second and third phases were interpreted according to a dissociative thermal inactivation model. These phases showed that lentil amine oxidase was reversibly-dissociated before the irreversible thermal inactivation. Although lentil amine oxidase is not a thermostable enzyme, its dimeric structure can form "conformational lock," conferring a structural tolerance to the enzyme against heat stress.

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“…Esta enzima aparece normalmente na forma de dímeros ou tetrâmeros (ROWLAND et al, 1994), sendo que as G6PDHs provenientes de Leuconostoc mesenteroides (Figura 1.1) e Saccharomyces cerevisiae são dímeros (COHEN, ROSEMEYER, 1969;ZAITZEVA et al, 1997). O pH ótimo de atividade da enzima é 7,8 e o ponto isoelétrico é 4,6 (OLIVE, LEVY, 1975;ISHAQUE et al, 1974;ROWLAND et al, 1994;COSGROVE et al, 1998).…”

Section: A Enzima Glicose-6-fosfato Desidrogenaseunclassified

Produção de glicose-6-fosfato desidrogenase de "Saccharomyces cerevisiae" geneticamente modificada através de processo descontínuo alimentado

Miguel¹

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Thermostability of yeast hexokinase and yeast glucose-6-phosphate dehydrogenase

Cited by 10 publications

References 5 publications

Quantitative Analysis of the High Temperature-induced Glycolytic Flux Increase in Saccharomyces cerevisiae Reveals Dominant Metabolic Regulation

Quantitative Analysis of the High Temperature-induced Glycolytic Flux Increase in Saccharomyces cerevisiae Reveals Dominant Metabolic Regulation

Conformational Lock and Dissociative Thermal Inactivation of Lentil Seedling Amine Oxidase

Produção de glicose-6-fosfato desidrogenase de "Saccharomyces cerevisiae" geneticamente modificada através de processo descontínuo alimentado

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