Non-linear slow-binding inhibition of Aerococcus viridans lactate oxidase by Cibacron Blue 3GA.
Lactate oxidase (LOD) was purified from cells of Aerococcus viridans by phase partitioning in Triton X-114 (TX-114), ammonium sulphate fractionation and FPLC ion exchange chromatography. The purification achieved from a crude extract of A. viridans was 32-fold with a 60% recovery of activity. The isolated enzyme was a true FMN-containing LOD in tetrameric form with a subunit molecular weight of 48,000. The KM for L-lactate was 175 microM, a 6-fold less value than described in the literature. Among the inhibitors tested, Cibacron Blue 3GA showed the lowest Ki. At low concentrations, Cibacron Blue 3GA behaved as a dye-, pH- and time-dependent inhibitor. A Dixon plot of the steady-state rate showed the time-dependent inhibition to be non-linear, contrary to that described for other slow-binding inhibitors. A model to explain this phenomenon was proposed. The model implies the binding of Cibacron Blue 3GA to the isomerised form of the initial enzyme-inhibition complex (E'I).
Streitenberger
S A
SA
Department of Biochemistry and Molecular Biology-A, Faculty of Biology, University of Murcia, Spain.
López-Mas
J A
JA
Sánchez-Ferrer
A
A
García-Carmona
F
F
D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification*; D008956:Models, Chemical; D008970:Molecular Weight; D017830:Octoxynol; D011485:Protein Binding; D013289:Streptococcaceae / Q000201:enzymology*; D013997:Time Factors; D014227:Triazines / Q000493:pharmacokinetics / Q000494:pharmacology*
D001051:Apoenzymes; D020035:Holoenzymes; D014227:Triazines; C012842:Cibacron Blue F 3GA; D017830:Octoxynol; D006899:Mixed Function Oxygenases; C021497:lactate 2-monooxygenase