Pyruvate kinase catalyses the final step in glycolysis and is one of three key control steps in the pathway. Here are kinetic parameters for wild type human liver pyruvate kinase (hL-PK) and two hL-PK missense mutants, (R401K and R401E) with phosphoenolpyruvate (PEP) as the substrate (0-50 mm). The kinetics were measured in the presence and absence of a fixed concentration (1.0 MM) of fructose-1,6- bisphosphate (F16BP). The affinity of F16BP for each protein was also measured. Kos Kos Kd With F16BP No F16BP F16BP (mm) (mm) (UM) Wild type 0.25 +0.05 25+ 3 0.05 0.01 R401K 0.22 +0.06 235 0.04 0.01 R401 2605 275 No binding (a) Draw on one set of axes what you predict the two kinetic plots (Vo vs. (PEP]) will look like for the wild type enzyme with and without F-1,6-BP. (3 marks) (b) Draw on a different set of axes what you predict the kinetic plots (Vo vs. (PEP]) will look like for the two mutant enzymes. (3 marks) (c) Assuming the mutations do not affect protein folding or stability, predict what functional impact the two mutations have had on hL-PK. Explain your answers in terms of allosteric conformational change, amino acid properties and the binding of substrates and allosteric modulators. (6 marks)