title: Substrate and pH-Dependent Kinetic Profile of 3-Mercaptopropionate Dioxygenase from Pseudomonas aeruginosa.
abstract: Thiol dioxygenases catalyze the synthesis of sulfinic acids in a range of organisms from bacteria to mammals. A thiol dioxygenase from the bacterium Pseudomonas aeruginosa oxidizes both 3-mercaptopropionic acid and cysteine, with a ∼70 fold preference for 3-mercaptopropionic acid over all pHs. This substrate reactivity is widened compared to other thiol dioxygenases and was exploited in this investigation of the residues important for activity. A simple model incorporating two protonation events was used to fit profiles of the Michaelis-Menten parameters determined at different pH values for both substrates. The pKs determined using plots of k(cat)/Km differ at low pH, but not in a way easily attributable to protonation of the substrate alone and share a common value at higher pH. Plots of k(cat) versus pH are also quite different at low pH showing the monoprotonated ES complexes with 3-mercaptopropionic acid and cysteine have different pKs. At higher pH, k(cat) decreases sigmoidally with a similar pK regardless of substrate. Loss of reactivity at high pH is attributed to deprotonation of tyrosine 159 and its influence on dioxygen binding. A mechanism is proposed by which deprotonation of tyrosine 159 both blocks oxygen binding and concomitantly promotes cystine formation. Finally, the role of tyrosine 159 was further probed by production of a G95C variant that is able to form a cysteine-tyrosine crosslink homologous to that found in mammalian cysteine dioxygenases. Activity of this variant is severely impaired. Crystallography shows that when un-crosslinked, the cysteine thiol excludes tyrosine 159 from its native position, while kinetic analysis shows that the thioether bond impairs reactivity of the crosslinked form.
authors.affiliation: Department of Chemistry and ‡Department of Biochemistry, University of Otago , P.O. Box 56, Dunedin 9054, New Zealand.
authors.forename: Matthias
authors.initials: M
authors.lastname: Fellner
authors.affiliation: Department of Chemistry and ‡Department of Biochemistry, University of Otago , P.O. Box 56, Dunedin 9054, New Zealand.
authors.forename: Sekotilani
authors.initials: S
authors.lastname: Aloi
authors.affiliation: Department of Chemistry and ‡Department of Biochemistry, University of Otago , P.O. Box 56, Dunedin 9054, New Zealand.
authors.forename: Egor P
authors.initials: EP
authors.lastname: Tchesnokov
authors.affiliation: Department of Chemistry and ‡Department of Biochemistry, University of Otago , P.O. Box 56, Dunedin 9054, New Zealand.
authors.forename: Sigurd M
authors.initials: SM
authors.lastname: Wilbanks
authors.affiliation: Department of Chemistry and ‡Department of Biochemistry, University of Otago , P.O. Box 56, Dunedin 9054, New Zealand.
authors.forename: Guy N L
authors.initials: GN
authors.lastname: Jameson
mesh_terms: D015097:3-Mercaptopropionic Acid / Q000737:chemistry* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D018360:Crystallography, X-Ray; D049308:Dioxygenases / Q000737:chemistry* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D015097:3-Mercaptopropionic Acid; D049308:Dioxygenases