Mutagenic, spectroscopic and functional investigations of pea cytosolic ascorbate peroxidase
thesisposted on 15.12.2014, 10:35 by Daniel Douglas. Turner
Recombinant pea cytosolic ascorbate peroxidase (wild-type rAPX) has been isolated and characterised using a variety of kinetic and spectroscopic techniques. Mutagenic replacement of single amino acid residues has been performed.;Electrospray mass spectrometry confirmed that the recombinant enzyme expressed without post-translational modification: the measured apoprotein MW (27192.8 +/- 0.8 Da) matched that deduced from the primary amino acid sequence (27192.8 Da). Wild-type rAPX was found to be spectroscopically identical to the native enzyme, i.e. bona fide enzyme isolated from Pisum Sativum L.; the steady- and transient-state kinetic profiles obtained are wholly consistent with that published in the literature.;Investigation of the pH-dependent haem-iron coordination geometry of the ferric resting-state of wild-type rAPX has confirmed the existence of novel six-coordinate low-spin 'acidic form', which was denoted by a peak at 1590 nm in the near-IR MCD spectrum. UV/visible spectra of the 'acidic form' of both H42A and wild-type rAPX were indistinguishable (lambda/nm = 410, 525.5, ~563sh & ~622sh), and the consensus of the pH-dependent data supports ligation of the ferric resting-state haem-iron by either an anomalous hydroxide or the distal arginine residue (Arg38) at low pH.;Phenylhydrazine-modification experiments suggest that site-directed replacement of Alai34 with proline does not measurably decrease the extent of haem exposure. Steady- and transient-state kinetics indicate that A134P oxidises ascorbate at a rate comparable (≈85%) to that of wild-type rAPX; a slight increase (≈3-fold) in the rate of phenolic oxidation was noted. These observations are consistent with the purported existence of two distinct substrate-binding sites in ascorbate peroxidase enzymes.;Quick-freeze EPR spectra have resolved a previously undetected tryptophan-based radical in Compound I*, the species that evolves---in the absence of ascorbate---from the initial intermediate (Compound I) formed by the reaction between wild-type rAPX and H2O2. The protein-based radical is probably not physiologically relevant.