posted on 2024-01-08, 06:29authored byHegne Pupart, Darja Vastšjonok, Tiit Lukk, Priit Väljamäe
Dye-decolorizing
peroxidases (DyPs) are heme-dependent
enzymes
that catalyze the oxidation of various substrates including environmental
pollutants such as azo dyes and also lignin. DyPs often display complex
non-Michaelis–Menten kinetics with substrate inhibition or
positive cooperativity. Here, we performed in-depth kinetic characterization
of the DyP of the bacterium Streptomyces coelicolor (ScDyPB). The activity of ScDyPB
was found to be dependent on its concentration in the working stock
used to initiate the reactions as well as on the pH of the working
stock. Furthermore, the above-listed conditions had different effects
on the oxidation of 2,2′-azino-di(3-ethyl-benzothiazoline-6-sulfonic
acid) (ABTS) and methylhydroquinone, suggesting that different mechanisms
are used in the oxidation of these substrates. The kinetics of the
oxidation of ABTS were best described by the model whereby ScDyPB exists as a mixture of two kinetically different
enzyme forms. Both forms obey the ping-pong kinetic mechanism, but
one form is substrate-inhibited by the ABTS, whereas the other is
not. Gel filtration chromatography and dynamic light scattering analyses
revealed that ScDyPB exists as a complex mixture
of molecules with different sizes. We propose that ScDyPB populations with low and high degrees of oligomerization have
different kinetic properties. Such enzyme oligomerization-dependent
modulation of the kinetic properties adds further dimension to the
complexity of the kinetics of DyPs but also suggests novel possibilities
for the regulation of their catalytic activity.