TITLE:
The Roles of pH in the Modification of Wild-Type Recombinant Phlebia radiata Manganese Peroxidase 3 Activities and Stability of Secondary Structures
AUTHORS:
Usenobong F. Ufot, Imeh J. Okop, Mfoniso P. Uko, Imekan I. Akpan, Khasim Cali, Monday I. Akpanabiatu
KEYWORDS:
Phlebia radiata, Manganese Peroxidase, pH-Dependence, Inactivation, Conformation
JOURNAL NAME:
American Journal of Molecular Biology,
Vol.12 No.4,
October
17,
2022
ABSTRACT: 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−),
a rapid alkaline transition occurs. Leaving the X− form of the enzyme
at pH 8.6, it slowly becomes converted to a third form of the enzyme Y−,
which returned to the original XH form of the enzyme at pH 6.7. Recovery of form
XH from form Y− 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−1,
and half-life (T1/2) = 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.