The pH dependence of the allosteric response of human liver pyruvate kinase to fructose-1,6-bisphosphate, ATP, and alanine

Abstract

The allosteric regulation of human liver pyruvate kinase (hL-PYK) by fructose-1,6-bisphosphate (Fru-1,6-BP; activator), ATP (inhibitor) and alanine (Ala; inhibitor) was monitored over a pH range from 6.5 to 8.0 at 37 degrees C. As a function of increasing pH, hL-PYK's affinity for the substrate phosphoenolpyruvate (PEP), and for Fru-1,6-BP decreases, while affinities for ATP and alanine slightly increases. At pH 6.5, Fru-1,6-BP and ATP elicit only small allosteric impacts on PEP affinity. As pH increases, Fru-1,6-BP and ATP elicit greater allosteric responses, but the response to alanine is relatively constant. Since the magnitudes of the allosteric coupling for ATP and for alanine inhibition are different and the pH dependences of these magnitudes are not similar, these inhibitors likely elicit their responses using different molecular mechanisms. In addition, our results fail to support a general correlation between pH dependent changes in effector affinity and pH dependent changes in the corresponding allosteric response.

Figure 1

Effects of Fru-1,6-BP, ATP, and Ala on the response of initial velocity to varying PEP concentration at pH 7.5 and 37°C. Lines represent the best fits to Equation 1 with the restrictions described in Material and Methods. Arrows indicate increasing effector concentration of Fru-1,6-BP (0, 0.000070, 0.00021, 0.0063, 0.0019, 0.0057, 0.017, and 0.051 mM), ATP (0, 0.51, 1.01, 2.02, 4.05, 8.1, 16.2, and 32.4 mM), or Ala (0, 0.782, 2.34, 7.03, 21, 63, and 570 mM).

Figure 2

K_app-PEP as a function of Fru-1,6-BP (◆), ATP (●), or Ala (□) at pH 7.5 and 37°C. K_app-PEP were obtained from fits to Equation 1. Lines represent the best fits of data as exemplified in Figure 1 to Equation 2. When error bars are not apparent, they are smaller than the data point symbols.

Figure 3

K_app-PEP as a function of Fru-1,6-BP, ATP, or Ala at and 37°C and pH 6.0 (●), 7.0 (□), 7.5 (▲), and 8.0 (○). The range of the y-axis is not consistent between graphs. Lines represent the best fits to Equation 2. When error bars are not apparent, they are smaller than the data point symbols.

Figure 4

Q_ax for the regulation by Fru-1,6-BP (▲), ATP (□), and Ala (●) as a function of pH. Q_ax values were obtained from fits of data as exemplified in Figure 3 to Equation 2. Lines represent data trends. When error bars are not apparent, they are smaller than the data point symbols.

Figure 5

K_a-PEP (●), K_ix-FBP (○), K_ix-ATP (□), and K_ix-Ala (△) as a function of pH. Affinity values were obtained from fits of the data in as exemplified in Figure 3 to Equation 2. Lines represent data trends. When error bars are not apparent, they are smaller than the data point symbols.