A source of ultrasensitivity in the glutamine response of the bicyclic cascade system controlling glutamine synthetase adenylylation state and activity in Escherichia coli

Biochemistry. 2011 Dec 20;50(50):10929-40. doi: 10.1021/bi201410x. Epub 2011 Nov 22.

Abstract

Glutamine synthetase (GS) activity in Escherichia coli is regulated by reversible adenylylation, brought about by a bicyclic system comprised of uridylyltransferase/uridylyl-removing enzyme (UTase/UR), its substrate, PII, adenylyltransferase (ATase), and its substrate, GS. The modified and unmodified forms of PII produced by the upstream UTase/UR-PII cycle regulate the downstream ATase-GS cycle. A reconstituted UTase/UR-PII-ATase-GS bicyclic system has been shown to produce a highly ultrasensitive response of GS adenylylation state to the glutamine concentration, but its composite UTase/UR-PII and ATase-GS cycles displayed moderate glutamine sensitivities when examined separately. Glutamine sensitivity of the bicyclic system was significantly reduced when the trimeric PII protein was replaced by a heterotrimeric form of PII that was functionally monomeric, and coupling between the two cycles was different in systems containing wild-type or heterotrimeric PII. Thus, the trimeric nature of PII played a role in the glutamine response of the bicyclic system. We therefore examined regulation of the individual AT (adenylylation) and AR (deadenylylation) activities of ATase by PII preparations with various levels of uridylylation. AR activity was affected in a linear fashion by PII uridylylation, but partially modified wild-type PII activated the AT much less than expected based on the extent of PII modification. Partially modified wild-type PII also bound to ATase less than expected based upon the fraction of modified subunits. Our results suggest that the AT activity is only bound and activated by completely unmodified PII and that this design is largely responsible for ultrasensitivity of the bicyclic system.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Enzyme Activation
  • Escherichia coli / enzymology
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism*
  • Glutamate-Ammonia Ligase / chemistry
  • Glutamate-Ammonia Ligase / genetics
  • Glutamate-Ammonia Ligase / metabolism*
  • Glutamine / metabolism*
  • Kinetics
  • Molecular Weight
  • Mutant Proteins / chemistry
  • Mutant Proteins / metabolism
  • Nucleotidyltransferases / chemistry
  • Nucleotidyltransferases / genetics
  • Nucleotidyltransferases / metabolism*
  • PII Nitrogen Regulatory Proteins / chemistry
  • PII Nitrogen Regulatory Proteins / genetics
  • PII Nitrogen Regulatory Proteins / metabolism*
  • Protein Processing, Post-Translational*
  • Protein Subunits / chemistry
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Signal Transduction

Substances

  • Escherichia coli Proteins
  • Mutant Proteins
  • PII Nitrogen Regulatory Proteins
  • Protein Subunits
  • Recombinant Proteins
  • Glutamine
  • Nucleotidyltransferases
  • glutamine-synthetase adenylyltransferase
  • regulatory protein uridylyltransferase
  • Glutamate-Ammonia Ligase