Systems-level analysis of mechanisms regulating yeast metabolic flux

TLDR

Cellular metabolic fluxes depend on enzyme activities and metabolite levels, yet conventional biochemical methods fail to capture how these concentrations vary across physiological states and regulate reactions. The study quantified enzyme, metabolite, and flux levels in 25 yeast steady states and modeled fluxes using Michaelis–Menten kinetics that incorporate enzyme, substrate, product, and regulator concentrations. The analysis uncovered three novel cross‑pathway regulatory interactions, including citrate‑mediated inhibition of pyruvate kinase that limits glycolysis under nitrogen limitation, and showed that substrate levels drive metabolic rates more strongly than enzymes, with metabolites exerting twice the impact of enzymes.

Abstract

Cellular metabolic fluxes are determined by enzyme activities and metabolite abundances. Biochemical approaches reveal the impact of specific substrates or regulators on enzyme kinetics but do not capture the extent to which metabolite and enzyme concentrations vary across physiological states and, therefore, how cellular reactions are regulated. We measured enzyme and metabolite concentrations and metabolic fluxes across 25 steady-state yeast cultures. We then assessed the extent to which flux can be explained by a Michaelis-Menten relationship between enzyme, substrate, product, and potential regulator concentrations. This revealed three previously unrecognized instances of cross-pathway regulation, which we biochemically verified. One of these involved inhibition of pyruvate kinase by citrate, which accumulated and thereby curtailed glycolytic outflow in nitrogen-limited yeast. Overall, substrate concentrations were the strongest driver of the net rates of cellular metabolic reactions, with metabolite concentrations collectively having more than double the physiological impact of enzymes.