Contribution of arginine-82 and arginine-86 to catalysis of RNases from Bacillus intermedius (binase)

FEBS Letters
G I YakovlevR W Hartley

Abstract

To elucidate the functional role of Arg82 and Arg86 in the enzyme activity of binase, the extracellular ribonuclease of Bacillus intermedius, we used site-directed mutagenesis. On cleavage of various substrates the catalytic activity of binase mutant Arg86 Ala is 2.7 x 10(3) - 7.7 x 10(3) times less than that of the native enzyme. The decrease in activity is determined preferentially by the decrease in the molecular rate constant kcat with a relatively small change of enzyme-substrate affinity, characterized by Km. This is the expected result if Arg86 acts to lower the energy of a transition state of the reaction. The replacement of Arg82 by Ala causes a 5-19-fold activity decrease, depending on the substrate. We propose that this residue does not have a direct catalytic function in the molecular mechanism of the binase action and that the activity decrease of binase on the replacement of Arg82 by alanine is mediated by the effect of Arg82 on the pK of catalytic residues.

References

Aug 1, 1976·Archives of Biochemistry and Biophysics·M Zabinski, F G Walz
Jan 1, 1979·FEBS Letters·G A AphanasenkoE S Severin
May 11, 1992·Nucleic Acids Research·A A SchulgaK G Skryabin
Jun 1, 1993·Journal of Biomolecular Structure & Dynamics·A A MakarovM P Kirpichnikov

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Citations

Feb 5, 2003·Bioorganic & Medicinal Chemistry Letters·Joshua J HigginRonald T Raines
Sep 23, 2003·Protein Science : a Publication of the Protein Society·Gennady I YakovlevAlexander A Makarov
May 29, 2007·Journal of the American Chemical Society·Frédéric AvenierFlorian Hollfelder

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