Pyruvate kinase activation volumes

Figure 12-20 Equilibria in pyruvate kinase reaction as studied by 31P NMR at 40.3 MHz, pH 8.0,15°C. (A-C) Equilibria with low enzyme in levels 15% 2H20. (A) nP NMR spectrum of 1.5 ml of reaction mixture PEP, 13.3 mM ADP, 14.1 mM MgCl2, 20 mM potassium Hepes buffer, 100 mM KC, 50 mM without enzyme. (B) Equilibrium mixture after the addition of 1 mg of pyruvate kinase to the reaction mixture. (C) Equilibrium after the addition of potassium pyruvate (final concentration of 200 mM) to the sample of the spectrum in (B). (D,E) Equilibrium with enzyme concentrations in excess of the substrates. Sample volumes 1.1 ml with 10% 2H20. (D) Equilibrium mixture set up with enzyme (2.8 mM active sites) 2.8 mM PEP 2.4 mM ADP 5.7 mM MgCl2 100 mM potassium Hepes 100 mM KC1. (E) Spectrum after the addition of 50 pi of 400 mM EDTA (pH readjusted to 8.0) to the sample of spectrum D. The EDTA removes metal ions, stopping the catalytic reactions and sharpening the resonances. From Nageswara Rao et al.685...

ATP inhibit nonspecific nucleotidases. Another way to prepare [a P] GTP Arfl for the GAP assay is to incubate Arfl with 25 ruM HEPES, pH 7.4, 100 mM NaCl, 3.5 mM MgCl2, 1 mM EDTA, 1 mM ATP, 1 fjM [a PJGTP (specific activity = 50,000-250,000 cpm/pmol), 25 ruM KCl, 1.25 U/ml pyruvate kinase, and 3 mM phosphoenolpyruvate. This buffer contains a GTP regenerating system. If using Arfl that has not been myristoy-lated, include 0.1% (w/v) Triton X-100. For myristoylated Arfl, use either micelles of 3 mM dimyristoylphosphatidylcholine and 0.1% cholate, pH 7.4 or use vesicles prepared by extrusion or sonication (see Chapter 15 of this volume. Assay and Properties of the Arf GAPs AGAPl, ASAPl, and ArfGAPl). 

Affinity Labeling. Affinity labeling of pyruvate kinase, which results in irreversible inactivation, is carried out by incubating the enzyme in a relatively small volume of buffer with the chosen concentration of DMPA. To assay for residual enzyme activity, small aliquots of pyruvate kinase are withdrawn at increasing time intervals and diluted successively into 0.05 M Tris buffer, then into the final assay cuvette containing 1 mM Mn + and 75 mM K+ but no inhibitor. An overall enzyme dilution of between 1500- and 6000-fold is easily achieved, thereby bringing the final concentration of DMPA in the assay mixture well below that which inhibits reversibly. 

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