Engineering and characterisation of novel protein covalent linkages in horseradish peroxidase (HRP) : effect on structure and function

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