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Indian Journal of Biochemistry and Biophysics (IJBB) >
IJBB Vol.47 [2010] >
IJBB Vol.47(3) [June 2010] >
| Title: | Mutational analysis for enzyme activity of mouse Gal 1,3GalNAc  2,3-sialyltransferase (mST3Gal I) |
| Authors: | Kim, Kyoung-Sook
Oh, Seoung-Un
Lee, Jai-Heon
Lee, Young-Choon |
| Keywords: | Sialyltransferase
Chemical modification
Site-directed mutagenesis |
| Issue Date: | Jun-2010 |
| Publisher: | CSIR |
| Abstract: | To determine
which amino acid residues are essential for the catalytic activity of mouse Gal1,3GalNAc 2,3-sialyltransferase (mST3Gal
I), chemical modification and site-directed mutagenesis were employed against
tryptophan and cysteine residues located in the predicted catalytic domain.
This enzyme was strongly inhibited by N-bromosuccinimide,
a specific blocking reagent for tryptophan residues, and the enzyme activity
was completely lost at 3 mM,
suggesting the involvement of tryptophan residues in the catalytic activity of
mST3Gal I. The N-ethylmaleimide, an
irreversible reagent for sulfhydryl group, significantly inhibited the enzyme
activity. Seven tryptophan and six cysteine residues conserved in the cloned Gal1,3GalNAc 2,3-sialyltransferases
were separately substituted into phenylalanine and serine, respectively. The enzymatic
activity assay for tryptophan mutants produced in COS cells showed a complete
abolishment of the activity in all of the mutants, except that W70F and W97F retained
about 60% and 40% activities of wild type, respectively. In the case of
cysteine mutants, no enzyme activity was observed like tryptophan mutants,
except for C139S. These results suggest that tryptophan and cysteine residues
conserved in ST3Gal I are critical for its activity. |
| Page(s): | 135-140 |
| ISSN: | 0975-0959 (Online); 0301-1208 (Print) |
| Source: | IJBB Vol.47(3) [June 2010]
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