Systematic Analysis of Flux Control in the Pentose Phosphate Pathway

Abstract

It has been known for the past half century that cancer cells show increased activity of the metabolic pathway glycolysis, though a consensus on the explanation for this phenomenon remains elusive. More recently a related pathway, the pentose phosphate pathway (PPP) has also come into focus as a crucial pathway for cancer cells. The PPP is separated into an oxidative and non-oxidative branch and produces ribose-5-phosphate and NADPH, a precursor for nucleotides and a reducing agent, respectively. How the PPP is regulated on an enzymatic level, how this regulation affects glycolysis, and which enzymes are primarily responsible are currently open questions. In order to answer these questions, I systematically overexpressed all enzymes in the PPP and used a combination of metabolomics and isotope-tracer methods to track metabolism. I found that overexpression of G6PD, proposed to be the rate-limiting step of the oxidative PPP, decreased flux through then non-oxidative PPP with no observable change to the oxidative PPP. G6PD overexpression also reduced glycolytic flux via accumulation of metabolic intermediates and increased NADPH pool size. A model of NADK inhibition did not explain this increased pool size. These findings provide a new understanding of the regulation of the PPP and the resulting changes to glycolysis.