Cooperativity and allosteric regulation of yeast diacylglycerol acyltransferase, DGA1

Abstract

The esterification of the Kennedy Pathway intermediate diacylglycerol generates the energy storage molecule, triglyceride. Alternatively, diacylglycerol can be used as a precursor for phospholipids. Therefore, diacylglycerol acyltransferase (DGAT), mainly mediated by DGA1 in S. cerevisiae, is a putative control point for the storage or structural utilization of acyl chains. Our in vitro DGAT assays using lro1are1are2 yeast microsomes indicated a distinct sigmoidal relationship between oleoyl‐CoA concentration and relative specific activity (Hill number = 3.0). This apparent cooperativity lead to our hypothesis that DGA1 is allosterically regulated. Subsequent in vitro DGAT assays included metabolites that, based on the regulation of other key enzymes in metabolism, are “metabolic indicators”. Elevated levels of ATP, NADH, acetyl‐CoA, and malonyl‐CoA are indicative of an energy‐rich cell. Elevated levels of AMP, ADP, and NAD+ are indicative of an energy‐poor cell. Initial experiments (n = 3) with estimated physiological concentrations of these metabolites found only 0.8 mM NAD+ to have an effect. The K0.5 was increased by about 20%. Such a direct connection between NAD+ levels and triglyceride synthetic enzymes has not, to our knowledge, been previously reported. Funding was provided by the Pennsylvania ‐ Delaware affiliate of the American Heart Association.