Benzoate Concentration and Cooperativity by a Substrate for Benzoate 1,2-dioxygenase from Benzoate-Degrading Rhodococcus Opacus 1CP

Abstract

We explored the effect of a change in substrate-benzoate (as sole carbon and energy source) concentration in growth medium on the activity of benzoate 1, 2-dioxygenase (BDO) of R.opacus 1CP cells, where BDO is the enzyme mediated the initial attack of benzoate. The activity of the enzyme was estimated by a change of respiration of whole freshly harvested bacterial cells (growth of the cells on benzoate) in response to injection of benzoate. It was shown that when concentration of growth substrate-benzoate decreased from 6 g/L to 250 mg/L, the curves of the dependency of the response rate to benzoate on the initial concentration of benzoate demonstrated that kinetics of the process changes from hyperbolic saturation kinetics, or typical Michaelis-Menten kinetics, to sigmoidal dependency of V on S. The semi saturation constant as a characteristic of the strength of substrate binding with BDO changed simultaneously. These changes were accompanied by the increase in the Hill coefficient from 1.02 up to 3.06, hence positive kinetic cooperativity by a substrate was observed for BDO of R.opacus 1CP cells. The influence of this type of cooperativity on viability of rhodococcus in natural environment and causes of the changes mentioned are discussed. It was hypothesized that an increase in substrate concentration in the medium for the growth of the bacterium not only stimulated synthesis of the inducible enzyme (BDO) in the cell but also led to the change in BDO conformation followed by the change in interaction between substrate-binding active sites of enzymes.

Evaluation of 3-Chlorobenzoate 1,2-Dioxygenase Inhibition by 2- and 4-Chlorobenzoate with a Cell-Based Technique

“…It consists of two different subunits. The sigmoidal dependency of V on S of benzoate and positive kinetic cooperativity by a substrate were observed for BDO of R. opacus 1CP cells [23]. It might be expected that the dependence of V on S for 3-CBA-1,2-DO of this culture would be sigmoidal.…”

Microbial Biosensor for Characterization of a Microorganism: A Review focusing on the Biochemical Activity of Microbial Cells

“…So, it was found that the concentration of the substrate, BA, in growth medium influenced the activity of BDO of Rhodococcus opacus 1CP. With increase of BA concentration, kinetics of the process catalyzed by BDO changes from the sigmoidal saturation Hill kinetics to the hyperbolic saturation Michaelis-Menten kinetics [35]. Using MMS, it was shown that induction led to classical Michaelis-Menten kinetics for the pair of Bacillus subtilis-fusaric acid [26].…”

Graphical Approach to Compare Concentration Constants of Hill and Michaelis-Menten Equations

“…A sigmoid dependence of the rate of enzymatic reaction on substrate concentration for allosteric enzymes can be described using empirical formula suggested by Hill [5]: close proximity to the amount of active sites [4]. In [9] it was shown that depending on the conditions of cultivation of the cells containing benzoate dioxygenase (allosteric enzyme), the Hill coefficient varied from 1 to 3. This coefficient could not be used in order to go from constant S 0.5 of Hill equation to Michaelis constant, K m .…”