title: Linear Free Energy Relationship Analysis of Transition State Mimicry by 3-Deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) Oxime, a DAHP Synthase Inhibitor and Phosphate Mimic.
abstract: 3-Deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase catalyzes an aldol-like reaction of phosphoenolpyruvate (PEP) with erythrose 4-phosphate (E4P) to form DAHP in the first step of the shikimate biosynthetic pathway. DAHP oxime, in which an oxime replaces the ketone, is a potent inhibitor, with Ki= 1.5 μM. Linear free energy relationship (LFER) analysis of DAHP oxime inhibition using DAHP synthase mutants revealed an excellent correlation between transition state stabilization and inhibition. The equations of LFER analysis were rederived to formalize the possibility of proportional, rather than equal, changes in the free energies of transition state stabilization and inhibitor binding, in accord with the fact that the majority of LFER analyses in the literature demonstrate nonunity slopes. A slope of unity, m = 1, indicates that catalysis and inhibitor binding are equally sensitive to perturbations such as mutations or modified inhibitor/substrate structures. Slopes <1 or >1 indicate that inhibitor binding is less sensitive or more sensitive, respectively, to perturbations than is catalysis. LFER analysis using the tetramolecular specificity constant, that is, plotting log(KM,MnKM,PEPKM,E4P/kcat) versus log(Ki), revealed a slope, m, of 0.34, with r2= 0.93. This provides evidence that DAHP oxime is mimicking the first irreversible transition state of the DAHP synthase reaction, presumably phosphate departure from the tetrahedral intermediate. This is evidence that the oxime group can act as a functional, as well as structural, mimic of phosphate groups.
authors.affiliation: Department of Chemistry & Chemical Biology and ‡Department of Biochemistry & Biomedical Sciences, McMaster University , 1280 Main Street West, Hamilton, Ontario L8S 4M1, Canada.
authors.forename: Naresh
authors.initials: N
authors.lastname: Balachandran
authors.affiliation: Department of Chemistry & Chemical Biology and ‡Department of Biochemistry & Biomedical Sciences, McMaster University , 1280 Main Street West, Hamilton, Ontario L8S 4M1, Canada.
authors.forename: Frederick
authors.identifier: 0000-0001-9487-6027
authors.initials: F
authors.lastname: To
authors.affiliation: Department of Chemistry & Chemical Biology and ‡Department of Biochemistry & Biomedical Sciences, McMaster University , 1280 Main Street West, Hamilton, Ontario L8S 4M1, Canada.
authors.forename: Paul J
authors.identifier: 0000-0003-3409-7907
authors.initials: PJ
authors.lastname: Berti
mesh_terms: D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D055162:Biocatalysis; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D018716:Molecular Mimicry; D009154:Mutation; D010091:Oximes / Q000737:chemistry*; D010728:Phosphoenolpyruvate / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012765:Shikimic Acid / Q000737:chemistry / Q000378:metabolism; D013403:Sugar Phosphates / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D010091:Oximes; D011993:Recombinant Fusion Proteins; D013403:Sugar Phosphates; C013469:3-deoxy-D-arabino-heptulosonate-7-phosphate; D012765:Shikimic Acid; C026959:erythrose 4-phosphate; D010728:Phosphoenolpyruvate; D010723:3-Deoxy-7-Phosphoheptulonate Synthase