Non-chemical proton-dependent steps prior to O2-activation limit Azotobacter vinelandii 3-mercaptopropionic acid dioxygenase (MDO) catalysis.
3-mercaptopropionate dioxygenase from Azotobacter vinelandii (Av MDO) is a non-heme mononuclear iron enzyme that catalyzes the O2-dependent oxidation of 3-mercaptopropionate (3mpa) to produce 3-sulfinopropionic acid (3spa). With one exception, the active site residues of MDO are identical to bacterial cysteine dioxygenase (CDO). Specifically, the CDO Arg-residue (R50) is replaced by Gln (Q67) in MDO. Despite this minor active site perturbation, substrate-specificity of Av MDO is more relaxed as compared to CDO. In order to investigate the relative timing of chemical and non-chemical events in Av MDO catalysis, the pH/D-dependence of steady-state kinetic parameters (kcat and kcat/KM) and viscosity effects are measured using two different substrates [3mpa and l-cysteine (cys)]. The pL-dependent activity of Av MDO in these reactions can be rationalized assuming a diprotic enzyme model in which three ionic forms of the enzyme are present [cationic, E((z+1)); neutral, E(z); and anionic, E((z-1))]. The activities observed for each substrate appear to be dominated by electrostatic interactions within the enzymatic active site. Given the similarity between MDO and the more extensively characterized mammalian CDO, a tentative model for the role of the conserved 'catalytic triad' is proposed.
Department of Chemistry & Biochemistry, College of Science, The University of Texas at Arlington, Arlington, TX 76019, USA.
Joshua K
JK
Crowell
Department of Chemistry & Biochemistry, College of Science, The University of Texas at Arlington, Arlington, TX 76019, USA.
Sinjinee
S
Sardar
Department of Chemistry & Biochemistry, College of Science, The University of Texas at Arlington, Arlington, TX 76019, USA.
Mohammad S
MS
Hossain
Department of Chemistry & Biochemistry, College of Science, The University of Texas at Arlington, Arlington, TX 76019, USA.
Frank W
FW
Foss
Department of Chemistry & Biochemistry, College of Science, The University of Texas at Arlington, Arlington, TX 76019, USA. Electronic address: bspierce@uta.edu.
Brad S
BS
Pierce
D015097:3-Mercaptopropionic Acid / Q000737:chemistry*; D001120:Arginine / Q000737:chemistry; D016948:Azotobacter vinelandii / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry*; D002384:Catalysis; D020134:Catalytic Domain; D002412:Cations; D003545:Cysteine / Q000737:chemistry; D050558:Cysteine Dioxygenase / Q000737:chemistry; D017666:Deuterium Oxide; D049308:Dioxygenases / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008968:Molecular Conformation; D010100:Oxygen / Q000737:chemistry*; D011522:Protons; D012997:Solvents / Q000737:chemistry; D055672:Static Electricity; D013379:Substrate Specificity; D014783:Viscosity
D001426:Bacterial Proteins; D002412:Cations; D011522:Protons; D012997:Solvents; D001120:Arginine; D015097:3-Mercaptopropionic Acid; D049308:Dioxygenases; D050558:Cysteine Dioxygenase; D017666:Deuterium Oxide; D003545:Cysteine; D010100:Oxygen
Non-heme mononuclear iron; Proton inventory; Solvent isotope effects; Thiol dioxygenase; Viscosity effects