Pyruvate kinase (from yeast)

Discussions of the regulation of glycolysis usually focus on phosphofructokinase. However, the step catalyzed by pyruvate kinase is also a highly exothermic step and part of a potential energy-wasteful cycle. Presumably, it is regulated to avoid that problem. In particular, when yeast is growing on a non-glucose or non-carbohydrate source, it will need to synthesize glucose by way of the gluconeogenic pathway. Certainly pyruvate kinase must be inhibited for this pathway to work. Under what conditions will it be inhibited? When there is a ready source of noncarbohydrate carbon (for example, an amino acid, like alanine) and plenty of ATP. Will this enzyme be subject to activation? If so, by what? ADP? AMP? How about fructose 1,6-bisphosphate? Why would this be a potential activator?

Partial isolation of pyruvate kinase

Ten g of active dry yeast is ground in a mortar with 5 mL washed sand in 20 mL of 0.1 M KP_i, pH 7.6, containing 5 mM MgCl₂, 5 mM EDTA, 2 mM PMSF (PMSF is only slightly soluble in water, so it must be dissolved first in a minimal volume of alcohol), and 40 mM 2-mercaptoethanol. The mortar and buffer are chilled before grinding and the grinding is done on ice. After grinding, the slurry is centrifuged at 20,000 g for 15 min. The supernatant is brought to 6% PEG 8000 by the addition of 40% PEG (w/w) while stirring. This mixture is then centrifuged at 10,000 g for 10 min. The pyruvate kinase is in the supernatant.

Pyruvate kinase activity

The reaction catalyzed by pyruvate kinase is not easily monitored directly. The two products can be measured, however, by various techniques. The simplest one is to use the enzyme, lactate dehydrogenase, along with NADH, to convert the product pyruvate to lactate with the concomitant oxidation of NADH to NAD⁺. This is accompanied by a decrease in the absorbance at 340 nm. The assay mixture contains 0.02-0.05 mL pyruvate kinase preparation, 25 mM KP_i, pH 7.6, 0.15 mM NADH, 0.4 mM PEP, 6 mM MgSO₄, 1.5 mM ADP, ~ 6 units LDH/mL. You will need to run a control to make sure there is no endogenous NADH oxidizing activity (or to be able to correct for it if there is). ADP and PEP can be made up as 10X or 20X stock solutions (neutralized). NADH should be made up fresh daily at 3 mM. Make only enough for one day's use. LDH should be made up from dry powder form to a 20X stock as well. It should be dissolved in a neutral buffer containing 20-40% glycerol so that it can be stored in the freezer between uses.

What are the kinetic constants for this enzyme (K_m and V_max)? Do activators and inhibitors seem to bind at the same or different sites? Are other glycolytic enzymes present in the preparation?