PMID: 2125820Dec 1, 1990Paper

Multiple replacements establish the importance of tyrosine-503 in beta-galactosidase (Escherichia coli)

Archives of Biochemistry and Biophysics
M Ring, R E Huber

Abstract

Tyr-503 of beta-galactosidase was specifically replaced with Phe, His, Cys, and Lys using site-directed mutagenesis. The normal enzyme and the substituted enzymes were purified. The activities of each of the substituted enzymes with o-nitrophenyl-beta-D-galactopyranoside (ONPG) and p-nitrophenyl-beta-D-galactopyronoside (PNPG) were very low and Y503K-beta-galactosidase was essentially inactive, showing that Tyr-503 is important for activity. The stability (including tetrameric stability) of the enzymes at 4 and 25 degrees C was essentially the same as that of the wild-type enzyme and the cleavage patterns on sodium dodecyl sulfate gels after protease action were unchanged. These studies thus indicate that Tyr-503 has no noticeable influence on stability under normal conditions. The substitutions for Tyr-503 had some small effects on the binding of both substrate and inhibitor. However, both kappa 2 (glycosidic bond cleavage rate) and kappa 3 (hydrolysis rate constant) were dramatically reduced. Each substitution except that of Lys (which can be explained by electrostatic effects) gave decreases in kappa 2 and kappa 3 of roughly the same magnitude regardless of whether the substitutions were conservative or not. This strongly im...Continue Reading

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Citations

Dec 3, 1999·Proteins·P VárnaiP D Lyne
Sep 5, 2002·Biotechnology and Bioengineering·Brett D MartinRenu Aggarwal
Mar 1, 1994·The International Journal of Biochemistry·R E HuberS K Khare
Aug 1, 1995·Nature Structural Biology·O el-KabbaniT G Flynn
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Jan 7, 2009·The Journal of Biological Chemistry·Masafumi HidakaShinya Fushinobu
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Jan 31, 2012·Applied and Environmental Microbiology·Joaquin Seras-FranzosoElena García-Fruitós
May 30, 2013·Molecular and Cellular Biology·Tek N LamichhaneRichard J Maraia
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Aug 2, 2001·The Journal of Biological Chemistry·N Ferrer-MirallesA Villaverde
Oct 3, 1999·Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire·R M PennerR E Huber
Apr 7, 1995·The Journal of Biological Chemistry·L ChenP B Høj

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