3. In your textbook the termi- nal enzyme catalyzing the ter- minal step of glycolysis is known as pyruvate kinase (PK). No indication is given that in mammalian tissues there are four different iso- forms of this enzyme. The four mammalian isoforms are known as PKL, PKR, PKM1 and PKM2. PKL is expressed in the liver, and PKR is ex- pressed in red blood cells. PKM1 and PKM2 are derived from altemative splicing of the PKM gene. PKM1 is a constitu- tively active isoform expressed Active Site A PKM2 Monomer B Fructose-1,6-Bisphosphate N Phe, Ala, Ser Binding Site PKM2 Tetramer "Activator" Binding Site in differentiated cells from various tissues. In contrast, PKM2 has low basal activity and is activated by an effector molecule fructose 1,6-bisphosphate (FBP). PKM2 is expressed in most proliferating cells. In addition, the regulation of PKM2 pyruvate kinase activity plays an essential role in cancer metabolism and is crucial for the growth and survival of cancer cells. Recently, PKM2 has been reported to function also as a nuclear protein kinase to regulate gene transcription and promote tumorigenesis. The diagram on the right illustrates the quaternary structure of the PKM2 tetramer and the binding sites of metabolic effector molecules. (a) In the laboratory, the catalytic activity of pyruvate kinase is measured by a continuous assay coupled to lactate dehy-drogenase because the reaction products of the PK reac- tion have no suitable absorption or fluorescence properties, in contrast to the properties of NADH and NAD*, as shown in the diagram on the right. Write both reactions with structural for- mulas as a coupled system naming all metabolites, cofactors, and enzymes. Under what conditions in the cell do these reac- tions occur and why is this important for continued turnover of glycolysis? Optical density NADH NAD+ wavelength (nm) Question #3, continued: (b) The diagram on the right illustrates Michaelis plots of wild type yeast pyruvate kinase and of a T403E mutant. The mutation hinders the binding of fructose-1,6-bisphosphate. The table below summarizes steady-state kinetic data of both the wild type and mutant pyruvate kinase in the absence and presence of 1 mM F-1,6-BPi. In the chart below, indicate which enzyme species in the Michaelis plots correspond to the indicated symbols. Base your answers on kinetic data in the table. Wild Type Kd (μM) no effector molecule F-1,6-BPi Kd (PEP) (μM) 1150 ΠΗ Kd (µM) T403E Kd (PEP) (μM) ΠΗ 3.7 960 2.7 10 50 1.0 >5400 830 2.6 1.00 0.80 Relative Velocity 0.40 €0.60 0.20 0.00 500 1000 1500 [PEP] μM 2000 2500 300 Indicate below corresponding wt enzyme or mutant with or without effector molecule × +

3. In your textbook the termi- nal enzyme catalyzing the ter- minal step of glycolysis is known as pyruvate kinase (PK). No indication is given that in mammalian tissues there are four different iso- forms of this enzyme. The four mammalian isoforms are known as PKL, PKR, PKM1 and PKM2. PKL is expressed in the liver, and PKR is ex- pressed in red blood cells. PKM1 and PKM2 are derived from altemative splicing of the PKM gene. PKM1 is a constitu- tively active isoform expressed Active Site A PKM2 Monomer B Fructose-1,6-Bisphosphate N Phe, Ala, Ser Binding Site PKM2 Tetramer "Activator" Binding Site in differentiated cells from various tissues. In contrast, PKM2 has low basal activity and is activated by an effector molecule fructose 1,6-bisphosphate (FBP). PKM2 is expressed in most proliferating cells. In addition, the regulation of PKM2 pyruvate kinase activity plays an essential role in cancer metabolism and is crucial for the growth and survival of cancer cells. Recently, PKM2 has been reported to function also as a nuclear protein kinase to regulate gene transcription and promote tumorigenesis. The diagram on the right illustrates the quaternary structure of the PKM2 tetramer and the binding sites of metabolic effector molecules. (a) In the laboratory, the catalytic activity of pyruvate kinase is measured by a continuous assay coupled to lactate dehy-drogenase because the reaction products of the PK reac- tion have no suitable absorption or fluorescence properties, in contrast to the properties of NADH and NAD*, as shown in the diagram on the right. Write both reactions with structural for- mulas as a coupled system naming all metabolites, cofactors, and enzymes. Under what conditions in the cell do these reac- tions occur and why is this important for continued turnover of glycolysis? Optical density NADH NAD+ wavelength (nm) Question #3, continued: (b) The diagram on the right illustrates Michaelis plots of wild type yeast pyruvate kinase and of a T403E mutant. The mutation hinders the binding of fructose-1,6-bisphosphate. The table below summarizes steady-state kinetic data of both the wild type and mutant pyruvate kinase in the absence and presence of 1 mM F-1,6-BPi. In the chart below, indicate which enzyme species in the Michaelis plots correspond to the indicated symbols. Base your answers on kinetic data in the table. Wild Type Kd (μM) no effector molecule F-1,6-BPi Kd (PEP) (μM) 1150 ΠΗ Kd (µM) T403E Kd (PEP) (μM) ΠΗ 3.7 960 2.7 10 50 1.0 >5400 830 2.6 1.00 0.80 Relative Velocity 0.40 €0.60 0.20 0.00 500 1000 1500 [PEP] μM 2000 2500 300 Indicate below corresponding wt enzyme or mutant with or without effector molecule × +

Biochemistry

6th Edition

ISBN:9781305577206

Author:Reginald H. Garrett, Charles M. Grisham

Publisher:Reginald H. Garrett, Charles M. Grisham

Chapter27: Metabolic Integration And Organ Specialization

Section: Chapter Questions

Problem 18P

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