N-linked glycopeptide mimetics as tools in kinetic, mechanistic and structural studies of peptide N:glycanase F : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University

Abstract

PNGases (Peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidases (E.C. 3.5.1.52) cleave the carbohydrate chains from the asparagine side chains of glycoproteins. They are widely used to deglycosylate N-linked glycoproteins and glycopeptides for analytical purposes. PNGase F from Flavobacterium meningosepticum is the best characterised of this class of enzymes but little is known so far about the biological significance or the catalytic mechanism of these intriguing enzymes. The substrate binding and cleavage mechanism of PNGase F has now been investigated. The first part of this work describes the synthesis of various novel N-linked glycopeptide mimetics which were then used in kinetic investigations with PNGase F. To facilitate kinetic studies at low substrate concentrations, a discontinuous HPLC based assay using a fluorescently labelled ovalbumin glycopeptide had to be developed. These experiments led to a better understanding of the structural requirements for substrate binding which will aid the future development of potent PNGase F inhibitors. In the second part of the thesis, a virtual N-linked glycopeptide from human lactoferrin was modelled into the active site region of PNGase F using molecular modelling techniques. This model has resulted in the proposal of a mechanism for catalysis that predicts an important role for Arginine 248, a residue that had previously not been considered part of the catalytic machinery. The model also provides a basis for explaining the substrate specificity of the enzyme. The mechanism is supported by kinetic studies with targeted PNGase F mutants. As a result of this study, new PNGase F mutants have been designed to test the current findings.