A Study of the Action of Lysozymes on Some New Synthetic Substrates and Inhibitors

Ballardie, Francis Westwood (1973) A Study of the Action of Lysozymes on Some New Synthetic Substrates and Inhibitors. PhD thesis, University of Glasgow.

Abstract

Hen egg white lysozyme has, in recent years been one of the most widely studied of enzymes, since its three-dimensional structure was determined by X-ray crystallography. From these studies, there have been proposed several mechanisms of action of the enzyme, at least three of which are possible. One object of this work was to determine which is the correct one. Despite the interest, only a handful of significant papers have appeared regarding the mechanism of action of the enzyme. This is because accurate kinetic investigation on the natural substrate is difficult or impossible, and all the synthetic substrates prepared prior to this work were very poor ones. The first object of this work was therefore to synthesise a good substrate whose hydrolysis could be followed easily. An obvious candidate was an aryl glycoside of NAG4, but there were considerable practical problems to overcome, since glycosides of NAG4 had not been previously prepared. The chromatographic separation, on a preparative scale, of the peracetates of NAG2 to NAG6 was achieved, and from these, some 2,4-dinitro, p-nitro, and 3,4-dinitrophenyl glycosides were synthesised. The increased specificity of the p-nitrophenyl glycosides and 3,4-dinitrophenyl glycosides on increasing the sugar chain length from NAG2 to NAG4 was observed , as expected. NAG4-beta-3,4 dinitrophenyl was a 250-fold better substrate for hen egg-white lysozyme than NAG2-beta-2,4 dinitrophenyl, the best synthetic substrate prepared prior to this work. NAG-beta-3,4-dinitrophenyl was less active than NAG6, and was used in unsuccessful attempts to detect a covalent intermediate in its hydrolysis. In an attempt to make even better substrates, the acetylated B -fluorides of NAG1, NAG2 and NAG4 were prepared. The former two were deacetylated, using a new deacetylation technique. These fluorides proved to have high spontaneous rates of hydrolysis. The NAG2-beta-fluoride proved to be a better substrate for lysozyme than NAG2-beta-2,4 dnp. The 2 methyl oxazoline derivative, formed by neighbouring group participation of the acetamido group, was shown to be present, by PMR during the methanolysis of NAG1-beta-fluoride. A series of inhibitors for the enzyme was studied, using NAG4-beta-3, 4-dinitrophenyl as substrate. These were the reducing sugars, NAG2 to NAG4, and their paranitrophenyl glycosides. Attempts were made to study the lactone inhibitors reported by Secemski. A comprehensive analysis of a full kinetic model of lysozyme reactions was carried out. The behaviour of many reactions was explained in terms of changes in non-productive complexes, and a distorted productive complex. Some important parameters for individual reactions were determined. NAG4-beta-3, 4 dinitrophenyl was also found to be a substrate for human milk, duck II and duck III lysozymes, and proved useful in the assay of hen-egg-white lysozymes.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Biochemistry
• Date of Award: 1973
• Depositing User: Enlighten Team
• Unique ID: glathesis:1973-78626
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 28 Feb 2020 12:09
• Last Modified: 28 Feb 2020 12:09
• URI: https://theses.gla.ac.uk/id/eprint/78626