Al-Fartusie, Falah Sumoon Daghal (2011) Engineering and characterisation of novel protein covalent linkages in horseradish peroxidase (HRP): effect on structure and function. Doctoral thesis (DPhil), University of Sussex.

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Abstract

Both mammalian and bacterial peroxidases contain novel covalent linkages. In
the former a sulfonium linkage to a critical Met residue is thought to modify the normal
planarity of the haem affecting the functional properties. Following on from the work of
Metcalfe et al., 2004 which showed that such links could be engineered in ascorbate
peroxidase, horseradish peroxidase (HRP) has been used as convenient model system to
try and understand the structural and electronic effects of engineered covalent linkages.
Previous work in the group (Cali, 2008) had shown that mutation of Ser167 to Met in
HRP-C* resulted in autocatalytic cross-linking on incubation with hydrogen peroxide.
In this thesis, two additional HRP variants, S167Y and S167W were studied and a new
novel structural linkage discovered.
The UV/Vis spectrum of the S167Y variant suggested a more 6-coordinate high
spin character. The molar extinction coefficients were markedly increased, 180 mM-1
cm-1 for S167Y and 135 mM-1 cm-1 for S167W, compared to 100 mM-1 cm-1 for the WT
enzyme, consistent with a more 6 coordinate high spin character normally seen in lignin
peroxidase. In contrast, the dissociation constant (Kd) of the S167W variant mutant for
the aromatic donor BHA was hardly affected, whilst that of the S167Y variant increased
two-fold relative to the WT, implying a significant perturbation of the aromatic donor
binding site and / or the associated haem-linked hydrogen bonding network.
After peroxide treatment the haem group of the S167Y variant could not be
extracted into acid butanone in contrast to the WT. Only a proportion of the haem could
be extracted even from the untreated S167Y variant, implying that a substantial fraction
of the protein had formed the haem-protein linkage during folding and purification.
These results were confirmed during reverse phase HPLC and MALDI-TOF / ESI mass
spectroscopy measurements. The haem and protein completely co-eluted in the case of
peroxide treated S167Y, while only ~50% of the haem was linked to the protein in the
untreated as isolated enzyme. The MALDI-TOF and ESI mass spectrum showed that
there was a large increase (614 Da) in the mass of the linked S167Y protein, compared
to that of the unlinked enzyme. Unlike the sulfonium linkage obtained earlier, treatment
with hydrogen peroxide was unnecessary to observe this increase. Interestingly, the
100% unlinked S167Y protein could only be isolated if enzyme was prepared in the
presence of an efficient peroxidase substrate as an antioxidant scavenger. It appears that
a Tyr residue at position 167 is highly reactive with respect of the haem vinyl side chain
forming a spontaneous covalent link not otherwise seen in nature.
Pre steady-state comparison of the intermediates has shown that Compound I
was formed essentially normally at near WT rates, however its stability was greatly
affected in the S167Y variant (linked or unlinked), the life time being decreased to
~0.04 s, compared to of the WT enzyme, where it was ~80 s. The substrate preference
of the cross-linked S167Y variant was also altered. Stopped-flow measurements of the
individual rate constants for the partial reactions of the catalytic cycle with luminol as
reducing substrate revealed an increase in the rate of reduction of Compound I to
Compound II (k2).
The X-ray crystal structure of S167Y variant was solved to 1.7 Å resolution and
the structure has been modelled and determined by x-ray crystallography. The x-ray
structure reveals an unanticipated linkage containing an additional ring structure bonded
to the engineered Tyr. ESI mass measurements supported this structure.

Item Type:	Thesis (Doctoral)
Schools and Departments:	School of Life Sciences > Biochemistry
Subjects:	Q Science > QD Chemistry > QD0241 Organic chemistry > QD0415 Biochemistry
Depositing User:	Library Cataloguing
Date Deposited:	20 Oct 2011 11:56
Last Modified:	21 Aug 2015 12:45
URI:	http://sro.sussex.ac.uk/id/eprint/7422

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