Mutational analysis for enzyme activity of mouse Galbeta1,3GalNAc alpha2,3-sialyltransferase (mST3Gal I).
To determine which amino acid residues are essential for the catalytic activity of mouse Galbeta1,3GalNAc alpha2,3-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 Galbeta1,3GalNAc alpha2,3-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.
Kim
Kyoung-Sook
KS
Brain Korea 21 Center for Silver-Bio Industrialization, Dong-A University, Busan 604-714, South Korea.
Oh
Seoung-Un
SU
Lee
Jai-Heon
JH
Lee
Young-Choon
YC
D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D020134:Catalytic Domain / Q000235:genetics; D002522:Chlorocebus aethiops; D017931:DNA Primers / Q000235:genetics; D066298:In Vitro Techniques; D051379:Mice; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012799:Sialyltransferases / Q000235:genetics* / Q000378:metabolism*; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
D017931:DNA Primers; D050505:Mutant Proteins; D011994:Recombinant Proteins; D012799:Sialyltransferases; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase