Study of some kinetic characteristics of the enzyme pyruvate kinase M2 (PKM2) purified from the sera of breast cancer patients

Abstract

Pyruvate Kinase M2 was purified through precipitating serum protein from breast cancer patients using 65% (NH₄)₂ SO₄, ion exchange chromatography was used CM-Cellulose to purify the enzyme pyruvate kinase M2, one protein peak was obtained from the washing step and another peak after recovery elution the results indicated that the enzyme activity 17.5U/mL, specific activity 145.8U/mg,  protein concentration  0.12mg/mL, yield 42% and the number of purification times 3 time. Purification results indicated pyruvate kinase M2 enzyme for gel filtration column sephadex G-200 to the emergence of only one protein band with high activity for the enzyme pyruvate kinase M2 the purifier has arrived 20U/mL and that specific activity 400U/mg, concentration protein 0.05 mg/L, yield 40%, and number times purification was 8 time. The optimal conditions were studied for the enzyme pyruvate kinase M2 purified from the sera of breast cancer patients, through the use of several concentrations of substrate phosphoenol pyruvate EPE , several valuable different from pH, and different temperatures, the results recorded values that were optimum concentration of the substrate 3mM,  pH7, optimal temperature 37^oC, and reaction time 30min, the stability of the PKM2 enzyme were also evaluated at different acid values and different temperatures, the enzyme PKM2 was more stability when the number pH 7-9,  and the enzyme recorded a remaining activity at a stable rate 80-100%,  it was stable and maintains its activity 100% at the temperature of 30-37^oC , and the enzyme recorded a remaining activity at a stable rate 28-100% up to temperature 52^oC. As it was account value Vmax  and Michaelis constant Km for enzyme  PKM2 purified from the sera of breast cancer patients, via the application of  relationship Lineweaver-Burke  plot  it was found that Vmax 20.5 and km 0.66.