Kinetic characteristics of L-lysine α- oxidase from Trichoderma cf. aureoviride Rifai VKM F-4268D: Substrate specificity and allosteric effects

Abstract

The present work aims to investigate the kinetic characteristics of homodimer enzyme L-lysine α-oxidase from Trichoderma cf. aureoviride Rifai VKM F-4268D, taking into account allosteric effects. The enzyme was first shown to reveal positive cooperativeness, h=2.05±0.15. Using additional opportunities of Hill coefficient the value of the Michaelis–Menten constant has been estimated, Km=1.015∙10−5М, indicating high strength of substrate binding to the active site of each subunit. High selectivity and absolute L-stereospecificity of the enzyme were shown. The inhibition of L-lysine conversion by non-cleavable lysine analogs as well as the reaction product was found out to take place. These effects have been evaluated only as the inhibition coefficients (%). A more detailed study of these inhibition effects was complicated because of the cooperativeness of enzyme subunits mentioned above. The kinetic scheme of L-lysine α-oxidase was proposed involving parallel-subsequent action of each of two subunits in the catalytic act.We think that the results obtained will be useful for studying the kinetic properties of other multi-subunit enzymes and improve understanding of the mechanisms of their action.

Allosteric Enzyme-Based Biosensors—Kinetic Behaviours of Immobilised L-Lysine-α-Oxidase from Trichoderma viride: pH Influence and Allosteric Properties

“…According to this point of view, catalytic schemes of LO reactions, such as those pointed out for LO from Trichoderma cf. aureoviride Rifai VKM F-4268D [ 16 ], can be hardly invoked in the present case, since they do not rely with any pH dependence of cooperativity, as is demonstrated in the present study. Since the MWC approach appears to be the best model describing the observed kinetics of LO from Trichoderma viride , some considerations and hypotheses need here to be postulated to tentatively explain the presence of two interconvertible conformational states of LO (the tense T and relaxed R states), their difference in affinity for Lys and their dependence on pH.…”

“…As pointed out elsewhere [ 15 , 16 ], kinetics studies of LO from Trichoderma viride and from Trichoderma cf. aureoviride Rifai VKM F-4268D showed a notable allosteric behaviour of these enzymes; in any case, those studies were performed at fixed pH values and the influence of pH on the cooperative behaviour of the enzyme was never explored.…”

“…The first kinetics studies evidencing the allosteric behaviour of LO [ 15 , 16 ] used the Hill equation to demonstrate the departure from the classical Michaelis-Menten approach and calculate the relevant Michaelis-Menten constant. Even in the present study, the Hill equation was preliminary used to show the dependence of the cooperativity on pH; a closer inspection of data compared to those reported in Figure 3 already evidenced a departure from the simple sigmoidal fitting.…”

“…In the present study, we will look further into the kinetic of immobilised LO from Trichoderma viride showing that its allosteric behaviour is strongly dependent on pH—the implications regarding the ionic form of Lys involved in the allosteric behaviour of the enzyme will be presented and discussed as well. Furthermore, we will show that the Monod-Wyman-Changeux (MWC) rather than the well-known Hill allosteric model, as previously used elsewhere [ 15 , 16 ], appears to be the best approach in describing the cooperative binding of LO. Finally, some considerations about the influence of the peculiar LO kinetics on biosensor operations and extracorporeal reactor device will be discussed.…”

“…For example, on increasing the working pH, an ever-increasing deviation from the classical Michaelis-Menten model of enzyme catalysis was reported [ 12 ], and unusual hydrodynamic behaviours were observed as well [ 12 , 13 ]; more recently, a kinetic study of immobilised LO from Trichoderma viride evidenced the allosteric behaviour of this enzyme [ 15 ], thus confirming the allosteric effects observed for LO from other sources (from Trichoderma cf. aureoviride Rifai VKM F-4268D), even if this latter kinetic studies were performed in solution [ 16 ]. Naturally, all these deviations from the classical model of enzyme catalysis require a thorough deepening if this enzyme need to be used in Lys assays and medical therapy.…”