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Human [alpha]-L-iduronidase : substrate synthesis and mechanistic analysis Nieman, Catharine E.
Abstract
Glycosidases are enzymes responsible for hydrolysis of glycosidic bonds. On the basis of sequence sirnilarities, glycosidases have been assigned to a series of families, one of which, Family 39, contains both β-D-xylosidases and α-L-iduronidases. However, the level of sequence similarity between these two enzymes is relatively low, calling into question mechanistic conclusions made about the iduronidases on the basis of results from xylosidases. By being assigned to a family of retaining glycosidases, human α -L-iduronidase (IDUA) is also predicted to operate with net retention of anomeric configuration, and sequence alignments suggest that E299 is the catalytic nucleophile. When 4- methylumbelliferyl- α -L-idopyranosiduronic acid was incubated with IDUA in the presence of 3 M methanol, ¹H NMR analysis of the resulting products revealed the presence of methyl a-L-idopyranosiduronic acid, and a complete absence of the β -methanolysis product, thus proving IDUA to be a retaining glycosidase. In order to identify the catalytic nucleophile, 5-fluoro- α -L-idopyranosyluronic acid fluoride (5F-IdoAF) was used to label the enzyme via trapping of the intermediate. MS analysis of a peptic digest of an assay mixture containing IDUA and 5F-IdoAF permitted the identification of the labeled peptide ²⁹¹ADTPIYNDEADPLVG³⁰⁵, having m/z = 893 ± 1. Further analysis revealed that the label was attached to E299, proving it to be the catalytic nucleophile. This, together with the proof of retention of anomeric configuration, supports the assignment of IDUA to Family 39. To further probe the mechanism of this enzyme, we required a series of aryl α-Liduronides of varying reactivities. Because the glycosylation chemistry of ido compounds is poorly understood, a methodology study was performed in which five glycosylation methods were tested for their ability to form the desired anomeric stereochemistry with a range of acceptors of varying pK[sub a]. The trichloroacetimidate method was found to produce the best results, and a series of aryl α -L-iduronides was synthesized. Kinetic parameters for their hydrolysis by IDUA were determined, and it was found that both kcai and k[sub cai]/K[sub m] were independent of leaving group pK[sub a]- This indicates that either glycosylation is rate-determining, with extremely efficient proton donation at the transition state, or that some other step, such as conformational change is ratedetermining.
Item Metadata
Title |
Human [alpha]-L-iduronidase : substrate synthesis and mechanistic analysis
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Creator | |
Publisher |
University of British Columbia
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Date Issued |
2000
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Description |
Glycosidases are enzymes responsible for hydrolysis of glycosidic bonds. On the
basis of sequence sirnilarities, glycosidases have been assigned to a series of families, one
of which, Family 39, contains both β-D-xylosidases and α-L-iduronidases. However, the
level of sequence similarity between these two enzymes is relatively low, calling into
question mechanistic conclusions made about the iduronidases on the basis of results
from xylosidases.
By being assigned to a family of retaining glycosidases, human α -L-iduronidase
(IDUA) is also predicted to operate with net retention of anomeric configuration, and
sequence alignments suggest that E299 is the catalytic nucleophile. When 4-
methylumbelliferyl- α -L-idopyranosiduronic acid was incubated with IDUA in the
presence of 3 M methanol, ¹H NMR analysis of the resulting products revealed the
presence of methyl a-L-idopyranosiduronic acid, and a complete absence of the β -methanolysis
product, thus proving IDUA to be a retaining glycosidase.
In order to identify the catalytic nucleophile, 5-fluoro- α -L-idopyranosyluronic acid
fluoride (5F-IdoAF) was used to label the enzyme via trapping of the intermediate. MS
analysis of a peptic digest of an assay mixture containing IDUA and 5F-IdoAF permitted
the identification of the labeled peptide ²⁹¹ADTPIYNDEADPLVG³⁰⁵, having m/z = 893
± 1. Further analysis revealed that the label was attached to E299, proving it to be the
catalytic nucleophile. This, together with the proof of retention of anomeric
configuration, supports the assignment of IDUA to Family 39.
To further probe the mechanism of this enzyme, we required a series of aryl α-Liduronides
of varying reactivities. Because the glycosylation chemistry of ido compounds
is poorly understood, a methodology study was performed in which five glycosylation
methods were tested for their ability to form the desired anomeric stereochemistry with a
range of acceptors of varying pK[sub a]. The trichloroacetimidate method was found to
produce the best results, and a series of aryl α -L-iduronides was synthesized. Kinetic
parameters for their hydrolysis by IDUA were determined, and it was found that both kcai
and k[sub cai]/K[sub m] were independent of leaving group pK[sub a]- This indicates that either
glycosylation is rate-determining, with extremely efficient proton donation at the
transition state, or that some other step, such as conformational change is ratedetermining.
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Extent |
5350162 bytes
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Genre | |
Type | |
File Format |
application/pdf
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Language |
eng
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Date Available |
2009-07-13
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Provider |
Vancouver : University of British Columbia Library
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Rights |
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
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DOI |
10.14288/1.0061396
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
2000-11
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Campus | |
Scholarly Level |
Graduate
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Aggregated Source Repository |
DSpace
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Rights
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.