Experiments related to intermediary metabolism and physical biochemistry. Travaux pratiques dans les domaines du métabolisme intermédiaire et de la biochimie physique.

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The effect of a G415R missense mutation in human pyruvate kinase isozyme M2 (hPM2) was investigated in this study in twofold: by first (1) determining how phenylalanine (Phe), oxalic acid (OA) and fructose-1,6-biphosphate (FBP) affected the specific activity of hPM2 and then (2) assessing how fructose-1,6-biphosphate and serine (Ser) influenced the stability of hPM2. A coupled-enzyme assay for which hPM2 catalyzed the conversion of phosphoenolpyruvate (PEP) to pyruvate which in turn was converted to lactate by lactate dehydrogenase (LDH) was used for the former while differential scanning fluorimetry was used for the latter. The specific activity of hPKM2 was established as 11.3±0.0085 U/mg for the wild type and 7.18±0.005 U/mg for the mutant (hPKM2G415R), providing support to past research. When exposed to 0.25mM of phenylalanine and 25mM of oxalic-acid the respective specific activities of hPKM2G415R were 0 U/mg and 13.1±0.014 U/mg. A decrease in the specific activity of hPKM2G415R in the presence of 1 mM of FBP was observed with increasing PEP concentration as expected. In regard to allosteric stability, the melting temperature (Tm) for hPKM2G415R without any effector was found to be 46.5°C but 52.8°C and 50.5°C in the presence of 5mM fructose-1,6-biphosphate and 5mM serine respectively. Using a shift in Tm(ΔTm) of 3°C to substantiate stability, both fructose-1,6-biphosphate and serine were deemed stabilizing ligands for hPKM2G415R – fructose-1,6-biphosphate being the stronger effector. This study confirmed previous reporting of hPKM2 being inhibited by phenylalanine and fructose-1,6-biphosphate, activated by oxalic-acid and stabilized by both fructose-1,6-biphosphate and serine. 

The goal of this research was to study the adaptation of E. Coli (37˚C) in the presence of 44 mg/L of hydrogen peroxide (H2O2) in an E. Coli culture. This required assessing: (1) cell growth and viability through cell culturing and bacterial plating; (2) the specific activity of lactate dehydrogenase through an enzyme assay and (3) the level of expression through RT–qPCR via relative quantification using SYBR-green. It was hypothesized that hydrogen peroxide would not only decrease the growth and viability of E. Coli cell but also the specific activity of lactate dehydrogenase from E. Coli and the level of gene expression in E. Coli. All hypotheses were supported. An 86% decrease in viable cells due to hydrogen peroxide exposure was observed following a one-week cell growth period. The specific activity of lactate dehydrogenase when converting pyruvate to lactate was found to be 1.72×10-5 U/ug and 1.25×10-5 U/ug for the control and hydrogen peroxide treated sample respectively, resulting in a 26% decrease in enzyme activity due to hydrogen peroxide. The respective cycle threshold (Ct) value for the control and H2O2 treated samples being 31.2 and 31.55 were found to be insignificant (Ct > 30). Nevertheless, a fold difference of 0.0616 confirmed hydrogen peroxide effect of reducing mRNA expression (94% decrease). Fluorescence was monitored as a function of temperature to evaluate the RT-qPCR results. The melting temperature of the house keeping gene (IdnT) and gene of interest (ldhA) were found to 81.3°C and 83°C. 

The effect of a G415R missense mutation in human pyruvate kinase isozyme M2 (hPM2) was investigated in this study in twofold: by first (1) determining how phenylalanine(Phe), oxalic acid (OA) and fructose-1,6-biphosphate (FBP) affected the specific activity of hPM2 and then (2) assessing how fructose-1,6-biphosphate and serine (Ser) influenced the stability of hPM2. A coupled-enzyme assay for which hPM2 catalyzed the conversion of phosphoenolpyruvate (PEP) to pyruvate which in turn was converted to lactate by lactate dehydrogenase (LDH) was used for the former while differential scanning fluorimetry was used for the latter. The specific activity of hPKM2 was established as 11.3±0.0085 U/mg for the wild type and 7.18±0.005 U/mg for the mutant (hPKM2G415R), providing support to past research. When exposed to 0.25mM of phenylalanine and 25mM of oxalic-acid the respective specific activities of hPKM2G415R were 0 U/mg and 13.1±0.014 U/mg. A decrease in the specific activity of hPKM2G415R in the presence of 1 mM of fructose-1,6-biphosphate was observed with increasing phosphoenolpyruvate concentration as expected. In regard to allosteric stability, the melting temperature (Tm) for hPKM2G415R without any effector was found to be 46.5°C but 52.8°C and 50.5°C in the presence of 5mM fructose-1,6-biphosphate and 5mM serine respectively. Using a shift in Tm(ΔTm) of 3°C to substantiate stability, both fructose-1,6-biphosphate and serine were deemed stabilizing ligands for hPKM2G415R – fructose-1,6-biphosphate being the stronger effector. This study confirmed previous reporting of hPKM2 being inhibited by phenylalanine and fructose-1,6-biphosphate, activated by oxalic-acid and stabilized by both fructose-1,6-biphosphate and serine. 

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