Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.453346
Title: Substrate distortion in lysozyme-substrate interactions
Author: Dearie, William McPherson
ISNI:       0000 0001 3421 235X
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1973
Availability of Full Text:
Access from EThOS:
Access from Institution:
Abstract:
The evidence supporting the hypothesis that substrate distortion towards the transition-state conformation in the lysozyme catalysed hydrolysis of glycosides makes an important contribution towards the rate enhancement observed for the enzymic hydrolysis is reviewed in the light of the currently held views on the mechanism of action of lysozyme and the known interactions of the enzyme with substrates and inhibitors. A series of oligosaccharides having the general structure (NAG)r-X, where n= 1-3, NAG= N-acetyl-D-glucosamine and X= an aryl glycoside were prepared. When X is an aryl glycoside of N-acetyl-D-xylosamine the effect of substrate distortion in the lysosyme catalysed hydrolysis should be eliminated. Therefore by comparison with other oligosaccharides for which substrate distortion could play a role in the enzymic hydrolysis, an estimation of this factor could be made. p-Nitrophenyl 2-acetamido-2-deoxy-beta-D-xylopyranoside was shown by N. M. R. to exhibit a dissociation constant and bound chemical shift value on association with lysozyme consistent with the sugar residue being bound in subsite C of the active cleft. This is in accord with the results obtained for the binding of other monosaccharide inhibitors having a 2-acetamido group, Nacetyl-D-glucosamine, methyl 2-acetamido-2-deoxy-alpha-D-gLucopyrano-side and p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside, confirming the strong interaction of the 2-acetamido group at subsite C. NAG-NAX-PNP was shown by N. M. R. to bind to lysozyme with the sugar residue proximal to the aglycone occupying subsite C of the active cleft, predicting that the compound binds to a considerable extent in the binding mode B, C. This binding mode was shown by N. M. R. to be present in the binding of NAG2, p-nitrophenyl beta-NAG2 and NAG-Glu-PNP to lysozyme. Within experimental error the same dissociation constant for the binding of NAG-NAX-PNP was obtained from fluorescence and inhibition studies. This compound was shown to bind more strongly to lysozyme than either NAG2 or p-nitrophenyl beta-NAG2 with a favourable energy difference of approximately 1Kcal/mole. Similarly from fluorescence and inhibition studies NAG2-NAX-PNP was shown to bind more strongly to lysozyme than either NAG3 or p-nitrophenyl beta-NAG3 by about 1Kcal/mole. The kinetics of the lysozyme catalysed hydrolysis of p-nitrophenyl beta-NAX, NAG-NAX-PNP, NAG2-NAX-PNP and NAG3-NAX-PNP were studied and compared with the lysozyme catalysed hydrolysis of the corresponding NAG glycosides. The rate of hydrolysis of the NAX compounds was found to be considerably slower than that of the NAG compounds. The upper limit of kcat /Km (apparent) found for the lysozyme catalysed hydrolysis of NAG3-NAX-PNP was 1,000 times less than kcat /km (apparent) found for p-nitrophenyl beta-NAG4 consistent with the hypothesis that substrate distortion is important in the lysozyme catalysed hydrolysis of glycosides. 3,4-dinitrophenyl beta-NAX, NAG-NAX-DNP and NAG2-NAX-DNP did not show any increase in the rate of hydrolysis in the presence of lysozyme over that found for the spontaneous hydrolysis, confirming the role of substrate distortion in the enzymic catalysis. The account that must be taken of the binding modes and strength of binding observed for the lower NAX containing oligosaccharides in the interpretation of the kinetic results is discussed. NAG-NAX-PNP, NAG2-NAX-PNP and NAG-Glu-PNP were shown to be competitive inhibitors of the lysozyme catalysed hydrolysis of 3,4-dinitro-phenyl beta-NAG4. The inhibition constant found for NAG-Glu-PNP differed from the dissociation constant for this compound found by N. M. R. The reasons for this difference are discussed in relation to the possible binding, modes available for this compound and the ability of the N. M. R. method to distinguish between different binding modes. p-Nitrophenyl 2-deoxy-beta-D-glucopyranoside was synthesized and fully characterised. Attempted transglycosylation reactions of this compound with lysozyme are discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.453346  DOI: Not available
Share: