Study of the thermal stability of D-amino acid oxidase from Trigonopsis variabilis reveals enzyme inactivation via multiple steps

Abstract

The thermal stability of a highly purified preparation of D-amino acid oxidase from Trigonopsis variabilis (TvDAO), which does not show microheterogeneity due to the partial oxidation of Cys-108, was studied based on dependence of temperature (20–60°C) and protein concentration (5–100 µmol L−1). The time courses of loss of enzyme activity in 100 mmol L−1 potassium phosphate buffer, pH 8.0, are well described by a formal kinetic mechanism in which two parallel denaturation processes, partial thermal unfolding and dissociation of the FAD cofactor, combine to yield the overall inactivation rate. Estimates from global fitting of the data revealed that the first-order rate constant of the unfolding reaction (ka) increased 104-fold in response to an increase in temperature from 20 to 60°C. The rate constants of FAD release (kb) and binding (k−b) as well as the irreversible aggregation of the apo-enzyme (kagg) were less sensitive to changes in temperature, their activation energy (Ea) being about 52 kJ mol−1 in comparison with an Ea value of 185 kJ mol−1 for ka. The rate-determining step of TvDAO inactivation switched from FAD dissociation to unfolding at high temperatures. The model adequately described the effect of protein concentration on inactivation kinetics. Its predictions regarding the extent of FAD release and aggregation during thermal denaturation were confirmed by experiments. TvDAO is shown to contain two highly reactive cysteines per protein subunit whose modification with 5,5′-dithio-bis (2-nitrobenzoic acid) was accompanied by inactivation. Dithiothreitol (1 mmol L−1) enhanced up to 10-fold the recovery of enzyme activity during ion exchange chromatography of technical-grade TvDAO. However, it did not stabilize TvDAO at all temperatures and protein concentrations, suggesting that deactivation of cysteines was not responsible for thermal denaturation.