Evidence That Does Not Support Pyruvate Kinase M2 (PKM2)-catalyzed Reaction as a Rate-limiting Step in Cancer Cell Glycolysis

Abstract

It has been recognized that the rate-limiting function of pyruvate kinase M2 (PKM2) in glycolysis plays an important role in distributing glycolytic intermediates for anabolic and catabolic purposes in cancer cells. However, after analysis of the catalytic capacity of PKM2 relative to other glycolytic enzymes, the regulation range of PKM2 activity, metabolic flux control, and thermodynamics, we suggest that the PKM2-catalyzed reaction is not a rate-limiting step in cancer cell glycolysis. Hexokinase and phosphofructokinase 1 (PFK1), the first and third enzyme along the pathway, are rate-limiting enzymes that limit the overall glycolytic rate, whereas PKM2 and lactate dehydrogenase, the last two enzymes in the pathway, are for the fast removal of upstream intermediates to prevent the obstruction of the pathway. The argument is in accordance with the catalytic capacity of glycolytic enzymes, regulation range of enzyme activities, metabolic flux control, and thermodynamics.During tumorigenesis, pyruvate kinase (PK) isotype in cells switches from pyruvate kinase M1 (PKM1) or pyruvate kinase L (PKL) to PKM2 (1, 2), suggesting that PKM2 plays a part in tumor initiation and development. PK catalyzes the last step of glycolysis and is commonly regarded as a rate-limiting enzyme. In cancer cells, PKM2 is considered as a rate-limiting enzyme, because its enzyme activity is sensitive to allosteric regulation (3, 4) and it catalyzes a thermodynamically favorable reaction. It is proposed that when it is allosterically inhibited, its catalytic rate is lower than the upstream glycolytic rate; hence, phosphoenolpyruvate (PEP) 4 and upstream glycolytic intermediate may accumulate and flow to anabolic pathways; when it is allosterically activated, its activity is higher than the upstream glycolytic rate, and the upstream glycolytic intermediate would flow to pyruvate, which is then converted to lactate or enters mitochondria for complete oxidation. There are several lines of evidence that support this hypothesis (1, 4 -11).However, the evidence to support PKM2 as a rate-limiting enzyme is not sufficient. If PKM2 is a rate-limiting enzyme, the following lines of evidence are required: (a) PKM2 catalyzes an irreversible reaction; (b) its enzyme activity relative to other glycolytic enzymes is low; (c) its activity is sensitive to regulation; and (d) when its activity is allosterically activated, its catalytic rate should be higher than the upstream glycolytic rate and when its activity is allosterically inhibited, its catalytic rate should be lower than upstream glycolytic rate. So far, there is evidence of points a and c but no b and d.

Perturbation of phosphoglycerate kinase 1 (PGK1) only marginally affects glycolysis in cancer cells

“…Kinetically, the actual activities of pyruvate kinase in control and PGK1knockdown cells (Supplementary table 1) were comparable and they were 1 to 2 orders of magnitude higher than the glycolytic rate, sufficiently to drive the upstream intermediates to pyruvate. The results were consistent with our previous study concerning the flux control of glycolysis in cancer cells with respect to the kinetics and thermodynamics of this enzyme (48).…”

“…In vitro Cell-free system model for glycolysis Previously we had described an in vitro cellfree system as a glycolysis model (48,49). We used the reaction buffer containing 200 mM HEPES, 0.5 mM EDTA , 100 mM KCl, 5 mM MgCl 2 , 5 mM Na 2 HPO 4 , 4 mM ADP, 1.5 mM ATP, 5 mM glucose, 0.1 mM NADH and 2 mM NAD for this glycolysis system.…”

Thermodynamic constraints on the regulation of metabolic fluxes

“…2B). We find that the relationship between reaction free energy changes and flux control coefficients calls into question the longstanding hypothesis that reactions with the most negative free energy changes, such as the reactions catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase in glycolysis, serve as rate-limiting steps of a pathway (35)(36)(37)(38). Although previous work in metabolic control analysis has provided the theoretical framework to study the role of thermodynamics in the regulation of metabolic fluxes (34), this hypothesis is still widely accepted (39 -41).…”

The multifaceted role of autophagy in cancer and the microenvironment

“…Cancer cells typically metabolize glucose to lactate, even when sufficient oxygen is present to support oxidative phosphorylation, a phenomenon known as the Warburg effect . Of note, pyruvate kinase (PKM2) is the final enzyme in the glycolytic pathway that controls the glycolytic flux and is therefore important for preventing accumulation of glycolytic intermediates . In cancer, PKM2 breakdown via CMA is increased, whereby reduced PKM2 associates with an accumulation of glycolytic intermediates that are rerouted toward branching biosynthetic pathways to support cancer growth .…”