title: Interaction between L-aspartic acid and L-asparaginase from Escherichia coli: binding and inhibition studies.
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 microM. 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 microM 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 approximately 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 beta-aspartyl enzyme intermediate.
authors.affiliation: Laboratory of Pharmacology and Experimental Therapeutics, National Cancer Institute, Bethesda, Maryland 20892.
authors.forename: H N
authors.initials: HN
authors.lastname: Jayaram
authors.forename: D A
authors.initials: DA
authors.lastname: Cooney
authors.forename: C Y
authors.initials: CY
authors.lastname: Huang
mesh_terms: D001215:Asparaginase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001216:Asparagine / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D011485:Protein Binding
chemical_list: D046911:Macromolecular Substances; D001216:Asparagine; D001215:Asparaginase