Papers by Ricardo Alarcon
New Perspectives Quarterly, 2008
Febs Journal, 2005
To examine the interaction of human arginase II (EC 3.5.3.1) with substrate and manganese ions, t... more To examine the interaction of human arginase II (EC 3.5.3.1) with substrate and manganese ions, the His120Asn, His145Asn and Asn149Asp mutations were introduced separately. About 53% and 95% of wild-type arginase activity were expressed by fully manganese activated species of the His120Asn and His145Asn variants, respectively. The Km for arginine (1.4–1.6 mm) was not altered and the wild-type and mutant enzymes were essentially inactive on agmatine. In contrast, the Asn149Asp mutant expressed almost undetectable activity on arginine, but significant activity on agmatine. The agmatinase activity of Asn149Asp (Km = 2.5 ± 0.2 mm) was markedly resistant to inhibition by arginine. After dialysis against EDTA, the His120Asn variant was totally inactive in the absence of added Mn2+ and contained < 0.1 Mn2+·subunit−1, whereas wild-type and His145Asn enzymes were half active and contained 1.1 ± 0.1 Mn2+·subunit−1 and 1.3 ± 0.1 Mn2+·subunit−1, respectively. Manganese reactivation of metal-free to half active species followed hyperbolic kinetics with Kd of 1.8 ± 0.2 × 10−8 m for the wild-type and His145Asn enzymes and 16.2 ± 0.5 × 10−8 m for the His120Asn variant. Upon mutation, the chromatographic behavior, tryptophan fluorescence properties (λmax = 338–339 nm) and sensitivity to thermal inactivation were not altered. The Asn149→Asp mutation is proposed to generate a conformational change responsible for the altered substrate specificity of arginase II. We also conclude that, in contrast with arginase I, Mn2+A is the more tightly bound metal ion in arginase II.
Archives of Biochemistry and Biophysics, 2004
The interaction of Escherichia coli agmatinase (EC 3.5.3.11) with the substrate guanidinium group... more 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 K m value for agmatine (from 1.1 ± 0.2 mM to 6.3 ± 0.3 mM) and the K i value for competitive inhibition by Gdn + (from 15.0 ± 0.1 mM to 44.2 ± 2.1 mM), but the K i value for putrescine inhibition (2.8 ± 0.2 mM) was not altered. The tryptophan fluorescence properties (k max = 342 nm) and circular dichroism spectra were not significantly altered by the Glu274 fi 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.
Physical Review Letters, 1999
We report on a measurement of the tensor-analyzing power T20 in elastic electron-deuteron scatter... more We report on a measurement of the tensor-analyzing power T20 in elastic electron-deuteron scattering in the range of four-momentum transfer from 1.8 to 3.2 fm-1. Electrons of 704 MeV were scattered from a polarized deuterium internal target. The tensor polarization of the deuterium nuclei was determined with an ion-extraction system, allowing an absolute measurement of T20. The data are described well by a non-relativistic calculation that includes the effects of meson-exchange currents.
International Journal of Food Engineering, 2009
... Use of Cassava Peel as Carbon Source for Production of Amylolytic Enzymes by Aspergillus nive... more ... Use of Cassava Peel as Carbon Source for Production of Amylolytic Enzymes by Aspergillus niveus Tony Marcio Silva∗ Ricardo Fernandes Alarcon Andre Ricardo de Lima Damasio Michele Michelin∗∗ Alexandre Maller Douglas C. Masui ...
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Papers by Ricardo Alarcon