Dataset Information

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.

SUBMITTER: Ebrecht AC

PROVIDER: S-EPMC5474683 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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

Ebrecht Ana C AC Solamen Ligin L Hill Benjamin L BL Iglesias Alberto A AA Olsen Kenneth W KW Ballicora Miguel A MA

Frontiers in chemistry 20170619

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 ...[more]

PMID: 28674689

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Project description:Although SAT (serine acetyltransferase) of Escherichia coli, which catalyses the first step in cysteine synthesis, proceeds via a random-order ternary complex reaction mechanism [Hindson and Shaw (2003) Biochemistry 42, 3113-3119], it has been suggested that the nearly identical enzyme from Salmonella typhimurium might involve an acetyl-enzyme intermediate [Leu and Cook (1994) Protein Peptide Lett. 1, 157-162]. In this study the alternative acetyl acceptor threonine and the alternative acyl donor, propionyl-CoA were used to further investigate the reaction mechanism of SAT from E. coli. Steady-state kinetic data and dead-end inhibition studies were again diagnostic of a random-order ternary complex reaction mechanism for alternative substrates. Since earlier kinetic studies with SAT from S. typhimurium suggested that cysteine competes with acetyl-CoA for binding, rather than serine with which it is isostructural, the specificity of the serine-binding pocket was assessed with three substrate mimics; beta-hydroxypropionic acid, glycine and ethanolamine. The data show that SAT interacts productively with the amino and hydroxymethyl moieties of serine, whereas the carboxyl group provides an essential contribution to binding strongly, supporting a view that cysteine will interact productively at the serine-binding site. Furthermore, since the hydroxymethyl contact region of the serine-binding site appears able to accommodate the methylene and acetyl moeties of threonine and O -acetyl-serine respectively, the site is unlikely to provide obligatory short-range contacts with the hydroxyl group of serine, a prerequisite for exclusion of cysteine. Such a proposal is supported by the results of micro-calorimetric studies which show that cysteine competes with serine for binding to SAT rather than with CoA. It follows that tight binding of cysteine at the serine-binding site near the catalytic centre may be the effector of a substantial reduction in the affinity of SAT for CoA, yielding the observed pattern of steady-state inhibition and the mechanism by which cysteine mediates effective end-product control of its synthesis.

Project description:BACKGROUND:ADP-glucose pyrophosphorylase (AGPase), which catalyses a rate limiting step in starch synthesis, is a heterotetramer comprised of two identical large and two identical small subunits in plants. Although the large and small subunits are equally sensitive to activity-altering amino acid changes when expressed in a bacterial system, the overall rate of non-synonymous evolution is approximately 2.7-fold greater for the large subunit than for the small subunit. Herein, we examine the basis for their different rates of evolution, the number of duplications in both large and small subunit genes and document changes in the patterns of AGPase evolution over time. RESULTS:We found that the first duplication in the AGPase large subunit family occurred early in the history of land plants, while the earliest small subunit duplication occurred after the divergence of monocots and eudicots. The large subunit also had a larger number of gene duplications than did the small subunit. The ancient duplications in the large subunit family raise concern about the saturation of synonymous substitutions, but estimates of the absolute rate of AGPase evolution were highly correlated with estimates of omega (the non-synonymous to synonymous rate ratio). Both subunits showed evidence for positive selection and relaxation of purifying selection after duplication, but these phenomena could not explain the different evolutionary rates of the two subunits. Instead, evolutionary constraints appear to be permanently relaxed for the large subunit relative to the small subunit. Both subunits exhibit branch-specific patterns of rate variation among sites. CONCLUSION:These analyses indicate that the higher evolutionary rate of the plant AGPase large subunit reflects permanent relaxation of constraints relative to the small subunit and they show that the large subunit genes have undergone more gene duplications than small subunit genes. Candidate sites potentially responsible for functional divergence within each of the AGPase subunits were investigated by examining branch-specific patterns of rate variation. We discuss the phenotypes of mutants that alter some candidate sites and strategies for examining candidate sites of presently unknown function.

Dataset Information

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

Publications

Allosteric Control of Substrate Specificity of the <i>Escherichia coli</i> ADP-Glucose Pyrophosphorylase.

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