Interaction between l-aspartic acid and l-asparaginase from escherichia coli: Binding and inhibition studies

Hiremagalur N. Jayaram, David A. Cooney, Charles Y. Huang

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Experiments using equilibrium dialysis and fluorescence quenching provided direct evidence that approximately four moles of L-aspartic acid were bound per mole of tetrameric L-asparaginase from Escherichia coli, with a dissociation constant on the order of 60-160 μM. In addition, a set of weaker binding sites with a dissociation constant in the millimolar range were detected. Kinetic studies also revealed that L-aspartic acid inhibited L-asparaginase competitively, with an inhibition constant of 80 μM at micromolar concentrations of L-asparagine; at millimolar concentrations of the amide, an increase in maximal velocity but a decrease in affinity for L-asparagine were observed. L-Aspartic acid at millimolar levels again displayed competitive inhibition. These and other observations suggest that L-aspartic acid binds not only to the active site but also a second site with lower intrinsic affinity for it. The observed "substrate activation" is most likely attributable to the binding of a second molecule of L-asparagine rather than negative cooperativity among the tight sites of the subunits of this tetrameric enzyme. Further support for L-aspartic acid binding to the active site comes from experiments in which the enzyme, when exposed to various group-specific reagents suffered parallel loss of catalytic activity and in its ability to bind L-aspartic acid. Different commercial preparations of Escherichia coli L-asparaginase were found to contain ∼ 2-4 moles of L-aspartic acid; these were incompletely removed by dialysis, but could be removed by transamination or decarboxylation. Efficiency of dialysis increased with increasing pH. Taken together, this set of results is consistent with the existence of a covalent βaspartyl enzyme intermediate.

Original languageEnglish (US)
Pages (from-to)151-161
Number of pages11
JournalJournal of Enzyme Inhibition and Medicinal Chemistry
Volume1
Issue number2
DOIs
StatePublished - 1986
Externally publishedYes

Fingerprint

Asparaginase
Aspartic Acid
Escherichia coli
Dialysis
Asparagine
Catalytic Domain
Enzymes
Decarboxylation
Amides
Quenching
Catalyst activity
Fluorescence
Experiments
Chemical activation
Binding Sites
Molecules
Kinetics
Substrates

Keywords

  • Binding
  • DONV
  • Inhibition
  • L-asparaginase
  • L-asparagine
  • L-Aspartic acid

ASJC Scopus subject areas

  • Drug Discovery
  • Pharmacology
  • Biochemistry
  • Molecular Medicine

Cite this

Interaction between l-aspartic acid and l-asparaginase from escherichia coli : Binding and inhibition studies. / Jayaram, Hiremagalur N.; Cooney, David A.; Huang, Charles Y.

In: Journal of Enzyme Inhibition and Medicinal Chemistry, Vol. 1, No. 2, 1986, p. 151-161.

Research output: Contribution to journalArticle

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