Brooks, DA, Fabrega, S, Hein, LK, Parkinson, EJ, P Durand, Yogalingam, G, Matte, U, Giugliani, R, Dasvarma, A, Eslahpazire, J, Henrissat, B, Mornon, JP, Hopwood, JJ and Lehn, P (2001), "Glycosidase active site mutations in human alpha-L-iduronidase.", Glycobiology, 11, 9: 741-50.
Abstract: Mucopolysaccharidosis type I (MPS I; McKusick 25280) results from a deficiency in
alpha-L-iduronidase activity. Using a bioinformatics approach, we have previously
predicted the putative acid/base catalyst and nucleophile residues in the
active site of this human lysosomal glycosidase to be Glu182 and
Glu299, respectively. To obtain experimental evidence supporting
these predictions, wild-type alpha-L-iduronidase and site-directed
mutants E182A and E299A were individually expressed in Chinese hamster
ovary-K1 cell lines. We have compared the synthesis, processing,
and catalytic properties of the two mutant proteins with wild-type
human alpha-L-iduronidase. Both E182A and E299A transfected cells
produced catalytically inactive human alpha-L-iduronidase protein at
levels comparable to the wild-type control. The E182A protein was
synthesized, processed, targeted to the lysosome, and secreted in a similar
fashion to wild-type alpha-L-iduronidase. The E299A mutant protein was
also synthesized and secreted similarly to the wild-type enzyme,
but there were alterations in its rate of traffic and proteolytic
processing. These data indicate that the enzymatic inactivity of the
E182A and E299A mutants is not due to problems of synthesis/folding,
but to the removal of key catalytic residues. In addition, we have
identified a MPS I patient with an E182K mutant allele. The E182K
mutant protein was expressed in CHO-K1 cells and also found to be
enzymatically inactive. Together, these results support the predicted role
of E182 and E299 in the catalytic mechanism of alpha-L-iduronidase
and we propose that the mutation of either of these residues would
contribute to a very severe clinical phenotype in a MPS I patient.
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