摘要
This investigation is aimed at understanding the specific role of pH and calcium ions on the activity and stability of wild-type recombinant Phlebia radiata manganese peroxidase 3 (rPr-MnP3). The pH-dependent cycle of reactions for rPr-MnP3 was evaluated by investigating time-dependent changes in the activity and electronic absorption spectrum of rPr-MnP3.The rPr-MnP3 had maximum efficacy (kcat/Km) for Mn (II) oxidation at pH 5.0 and 3.0 for oxidation of ABTS. Raising the pH of a solution of resting rPr-MnP3 from pH 6.7 (form XH) to pH 8.6 (form X<sup>−</sup>), a rapid alkaline transition occurs. Leaving the X<sup>−</sup> form of the enzyme at pH 8.6, it slowly becomes converted to a third form of the enzyme Y<sup>−</sup>, which returned to the original XH form of the enzyme at pH 6.7. Recovery of form XH from form Y<sup>−</sup> occurred through an intermediate Z form. The pH inactivation of rPr-MnP3 followed first-order kinetics. The rate of formation of XH from Z is pH-dependent and biphasic in nature, with measured rate constants (k) = 0.25 min<sup>−1</sup>, and half-life (T<sub>1/2</sub>) = 2.8 min. The pH-dependent properties observed may be indicative of a greater degree of conformational flexibility at rPr-MnP3 active site due to disruption of the haem-linked hydrogen-bonding network in the distal haem pocket. Calcium ions were observed to significantly stabilised the enzyme’s spectral features and reduce the loss of activity during the alkaline pH transition. Calcium ions enhance the recovery of the initial activity but cannot prevent the final time-dependent irreversible denaturation and aggregation.
This investigation is aimed at understanding the specific role of pH and calcium ions on the activity and stability of wild-type recombinant Phlebia radiata manganese peroxidase 3 (rPr-MnP3). The pH-dependent cycle of reactions for rPr-MnP3 was evaluated by investigating time-dependent changes in the activity and electronic absorption spectrum of rPr-MnP3.The rPr-MnP3 had maximum efficacy (kcat/Km) for Mn (II) oxidation at pH 5.0 and 3.0 for oxidation of ABTS. Raising the pH of a solution of resting rPr-MnP3 from pH 6.7 (form XH) to pH 8.6 (form X<sup>−</sup>), a rapid alkaline transition occurs. Leaving the X<sup>−</sup> form of the enzyme at pH 8.6, it slowly becomes converted to a third form of the enzyme Y<sup>−</sup>, which returned to the original XH form of the enzyme at pH 6.7. Recovery of form XH from form Y<sup>−</sup> occurred through an intermediate Z form. The pH inactivation of rPr-MnP3 followed first-order kinetics. The rate of formation of XH from Z is pH-dependent and biphasic in nature, with measured rate constants (k) = 0.25 min<sup>−1</sup>, and half-life (T<sub>1/2</sub>) = 2.8 min. The pH-dependent properties observed may be indicative of a greater degree of conformational flexibility at rPr-MnP3 active site due to disruption of the haem-linked hydrogen-bonding network in the distal haem pocket. Calcium ions were observed to significantly stabilised the enzyme’s spectral features and reduce the loss of activity during the alkaline pH transition. Calcium ions enhance the recovery of the initial activity but cannot prevent the final time-dependent irreversible denaturation and aggregation.
作者
Usenobong F. Ufot
Imeh J. Okop
Mfoniso P. Uko
Imekan I. Akpan
Khasim Cali
Monday I. Akpanabiatu
Usenobong F. Ufot;Imeh J. Okop;Mfoniso P. Uko;Imekan I. Akpan;Khasim Cali;Monday I. Akpanabiatu(Department of Biochemistry, Faculty of Biological Sciences, Akwa Ibom State University, Uyo, Nigeria;Department of Chemistry, Faculty of Biological Sciences, Akwa Ibom State University, Uyo, Nigeria;Department of Microbiology, Faculty of Biological Sciences, Akwa Ibom State University, Uyo, Nigeria;Department of Zoology, Faculty of Biological Sciences, Akwa Ibom State University, Uyo, Nigeria;Department of Chemical Engineering and Analytical Science, University of Manchester, Manchester, UK;Department of Biochemistry, Faculty of Sciences, University of Uyo, Uyo, Nigeria)
