Here we show that ultrasensitive allosteric activation of PEP carboxylase by FBP plays a major role in controlling central metabolic activity in E. coli. At the protein level, such regulation involves sensitization of PEP carboxylase to FBP by physiological concentrations of acetyl-CoA and aspartate. At the systems level, it enables accumulation of PEP upon glucose removal, which in turn provides an advantage in reinitiating glycolysis when glucose reappears.
A key question is whether the ultrasensitive nature of the regulation is truly required to achieve PEP accumulation. A simple mathematical model of glycolytic regulation demonstrates that the observed ultrasensitivity is a prerequisite for PEP showing inverse concentration changes to FBP (Supplementary Fig. 15
), and thus PEP accumulating when glycolytic flux decreases, as has been observed experimentally in E. coli
and other organisms30,31
. Like PEP carboxylase, the pyruvate kinase isozyme which is expressed in the presence of glucose (PykF) is activated by FBP32
. We anticipate that, in the presence of physiological metabolite concentrations, PykF will also respond ultrasensitively to FBP, rendering PEP accumulation a robust physiological response to glucose removal.
The effects of PEP carboxylase and pyruvate kinase regulation may be augmented by allosteric inactivation of phosphofructokinase by PEP33
: initial depletion of FBP and build-up of PEP will promote further inactivation of both enzymes, thereby locking glycolysis in an off state until a suitable glycolytic substrate reappears.
A putative advantage of allosteric regulation is speed34
. This seems paramount for shut off of PEP carboxylase and pyruvate kinase upon glucose removal, where termination of glycolytic egress must out race depletion of upstream glycolytic compounds. In contrast, in cases where enzymes in E. coli
are inactivated via covalent modification35
, there is not a corresponding need for fast regulation. For example, the central nitrogen metabolic enzyme glutamine synthetase is turned off by covalent modification upon ammonia upshift36
; in this case, the delay introduced by the covalent modification step may be advantageous, favoring uptake of a useful initial burst of nitrogen.
An unexpected feature of PEP carboxylase’s allosteric activation is its ability to produce switch-like flux control that overrides the effects of substrate levels. PEP carboxylase is a tetrameric enzyme, compromised of a four identical catalytic units37
. The same is true of PykF32
. A likely reason for the evolution of multimeric enzymes is to enable ultrasensitivity, to substrate38–42
, to feedback inhibitors in biosynthesis43,44
, or, as shown here, to feed forward activation in central carbon metabolism. The existence of such intrinsically ultrasensitive enzymes confers metabolism with substantial capabilities for self-regulation.