title: Dihydrodipicolinate synthase from Campylobacter jejuni: kinetic mechanism of cooperative allosteric inhibition and inhibitor-induced substrate cooperativity.
abstract: Dihydrodipicolinate synthase (DHDPS), an enzyme of the meso-diaminopimelate pathway of lysine biosynthesis, is essential for bacterial growth and is considered a target for novel antibiotics. We have studied DHDPS from Campylobacter jejuni for the first time, determining the kinetic mechanism of catalysis and inhibition with its natural allosteric feedback inhibitor (S)-lysine. The tetrameric enzyme is known to have two allosteric sites, each of which binds two molecules of lysine. The results suggest that lysine binds highly cooperatively, and primarily to the F form of the enzyme during the ping-pong mechanism. By applying graphical methods and nonlinear regression, we have discriminated between the possible kinetic models and determined the kinetic and inhibition constants and Hill coefficients. We conclude that (S)-lysine is an uncompetitive partial inhibitor with respect to its first substrate, pyruvate, and a mixed partial inhibitor with respect to its second substrate, (S)-aspartate-β-semialdehyde (ASA), which differs from the kinetic models for inhibition reported for DHDPS from other sources. The Hill coefficients for the binding of lysine to different forms of the enzyme are all greater than 2, suggesting that the two allosteric sites are not independent. It has been found that ASA binds cooperatively in the presence of (S)-lysine, and the cooperativity of binding increases at near-KM concentrations of pyruvate. The incorporation of Hill coefficients into the kinetic equations was crucial for determining the kinetic model for this enzyme.
authors.affiliation: Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, Saskatchewan, Canada, S7N 5C9.
authors.forename: Yulia V
authors.initials: YV
authors.lastname: Skovpen
authors.forename: David R J
authors.initials: DR
authors.lastname: Palmer
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D016123:Campylobacter jejuni / Q000201:enzymology* / Q000378:metabolism; D002384:Catalysis; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D008958:Models, Molecular; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D001224:Aspartic Acid; C006644:aspartic semialdehyde; D006836:Hydro-Lyases; C020040:4-hydroxy-tetrahydrodipicolinate synthase; D008239:Lysine