Allosteric Control of Substrate Specificity of the Escherichia coli ADP-Glucose Pyrophosphorylase

Abstract

The substrate specificity of enzymes is crucial to control the fate of metabolites to different pathways. However, there is growing evidence that many enzymes can catalyze alternative reactions. This promiscuous behavior has important implications in protein evolution and the acquisition of new functions. The question is how the undesirable outcomes of in vivo promiscuity can be prevented. ADP-glucose pyrophosphorylase from Escherichia coli is an example of an enzyme that needs to select the correct substrate from a broad spectrum of alternatives. This selection will guide the flow of carbohydrate metabolism toward the synthesis of reserve polysaccharides. Here, we show that the allosteric activator fructose-1,6-bisphosphate plays a role in such selection by increasing the catalytic efficiency of the enzyme toward the use of ATP rather than other nucleotides. In the presence of fructose-1,6-bisphosphate, the kcat/S0.5 for ATP was near ~600-fold higher that other nucleotides, whereas in the absence of activator was only ~3-fold higher. We propose that the allosteric regulation of certain enzymes is an evolutionary mechanism of adaptation for the selection of specific substrates.

Structural analysis reveals a pyruvate-binding activator site in the Agrobacterium tumefaciens ADP–glucose pyrophosphorylase

“…We recently found that pyruvate plays a relevant role in the regulation of the enzyme from E. coli (11,23). This was previously overlooked because its synergistic effect was only detected in the presence of the main activator, FBP (11,23). Possibly other enzymes in this family follow a similar trend, and the structural information provided in this work will serve for better predictions.…”

“…It is not clear how many enzymes from the ADP-Glc PPase family are activated by pyruvate, which is critical evolutionary information and therefore deserving of further investigation. We recently found that pyruvate plays a relevant role in the regulation of the enzyme from E. coli (11,23). This was previously overlooked because its synergistic effect was only detected in the presence of the main activator, FBP (11,23).…”

Functional Characterisation of Three Glycine N-Acyltransferase Variants and the Effect on Glycine Conjugation to Benzoyl–CoA

“…However, with the tenfold lower k cat , this increased potency is unlikely to offer significant advantages due to an increase in the population of non-productive binding conformations [ 65 ]. Allosteric regulation of certain enzymes is an evolutionary mechanism of adaptation for the selection of specific substrates because the enzyme specificity for substrates controls metabolic flow by sorting metabolites into distinct paths [ 66 ]. The glycine conjugation pathway maintains a delicate balance in CoA levels within the mitochondria [ 21 ] and this might explain why deleterious variants such as the 156Asn > Ser,199Arg > Cys haplotype are maintained at very low frequencies in the population.…”

Regulatory Properties of the ADP-Glucose Pyrophosphorylase from the Clostridial Firmicutes Member Ruminococcus albus

“…We further explored the regulation of the R. albus ADP-Glc PPase forms by analyzing changes produced by the effectors on the substrate kinetics, determining the enzyme catalytic efficiency ratio V max /S 0.5 ; equivalent to k cat /K m for hyperbolic kinetics (11,(16)(17)(18) for comparative analysis. As detailed in Table 1, allosteric effectors modified V max and/or S 0.5 values to different extents.…”