Kinetic studies and site-directed mutagenesis of Escherichia coli agmatinase. A role for Glu274 in binding and correct positioning of the substrate guanidinium group

Archives of Biochemistry and Biophysics
Nelson CarvajalV López

Abstract

The interaction of Escherichia coli agmatinase (EC 3.5.3.11) with the substrate guanidinium group was investigated by kinetic and site-directed mutagenesis studies. Putrescine and guanidinium ions (Gdn+) were slope-linear, competitive inhibitors with respect to agmatine and their bindings to the enzyme were not mutually exclusive. By site-directed mutagenesis, the E274A variant exhibiting about 1-2% of wild-type activity was obtained. Mutation produced a moderate, but significant, increase in the Km value for agmatine (from 1.1 +/- 0.2 mM to 6.3 +/- 0.3 mM) and the Ki value for competitive inhibition by Gdn+ (from 15.0 +/- 0.1 mM to 44.2 +/- 2.1 mM), but the Ki value for putrescine inhibition (2.8 +/- 0.2 mM) was not altered. The tryptophan fluorescence properties (lambdamax = 342 nm) and circular dichroism spectra were not significantly altered by the Glu274 --> Ala mutation. The dimeric structure of the enzyme was also maintained. We conclude that Glu274 is involved in binding and positioning of the guanidinium moiety of the substrate for efficient catalysis. A kinetic mechanism involving rapid equilibrium random release of products is proposed for E. coli agmatinase.

References

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Citations

Jul 28, 2010·Applied Microbiology and Biotechnology·Jens Schneider, Volker F Wendisch
Aug 26, 2010·Current Opinion in Critical Care·Joanne RichardsonBrian H Cuthbertson
Apr 16, 2021·PloS One·Iva ChitrakarJarrod B French
May 6, 2021·International Journal of Molecular Sciences·Pablo MaturanaElena Uribe
Jan 31, 2015·Journal of Inorganic Biochemistry·Matías QuiñonesElena Uribe

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