Papers by Mario L. Calcagno
Journal of Steroid Biochemistry, 1985
Archives of Biochemistry and Biophysics, 1989
Methylation of glucosamine-6-phosphate isomerase deaminase (2-amino-%deoxy-Dglucose-6-phosphate k... more Methylation of glucosamine-6-phosphate isomerase deaminase (2-amino-%deoxy-Dglucose-6-phosphate ketol-isomerase, deaminating, or glucosamine-6-phosphate deaminase, EC 5.3.1.10), from Escherichia coli produces a modified protein having two alkylated sulfhydryls per each polypeptide chain. The enzyme is still active and allosteric, but exhibits a lower homotropic cooperativity and its V,,,ax/Et,,tal is almost exactly half that of the native enzyme. Arsenite produces comparable kinetic changes that can be reversed with ethanedithiol but not with 2-thioethanol or dialysis. Thiols can be oxidized by molecular oxygen using the (l,lO-phenanthroline),-Cu(I1) complex as catalyst; the enzyme obtained no longer has titrable SH groups with 5,5'-dithiobis(2-nitrobenzoic acid) and displays kinetic behavior similar to that of the other chemically modified forms of the deaminase using monofunctional or bifunctional reagents. The results reported indicate that the involved sulfhydryls are vicinal groups, and are located in a region of the molecule that moves as a whole in the allosteric transition.
Archives of Biochemistry and Biophysics, 2005
Glucosamine-6-phosphate deaminase from Escherichia coli (EC 3.5.99.6) is an allosteric enzyme, ac... more Glucosamine-6-phosphate deaminase from Escherichia coli (EC 3.5.99.6) is an allosteric enzyme, activated by N-acetylglucosamine 6-phosphate, which converts glucosamine-6-phosphate into fructose 6-phosphate and ammonia. X-ray crystallographic structural models have showed that Arg172 and Lys208, together with the segment 41-44 of the main chain backbone, are involved in binding the substrate phospho group when the enzyme is in the R activated state. A set of mutants of the enzyme involving the targeted residues were constructed to analyze the role of Arg172 and Lys208 in deaminase allosteric function. The mutant enzymes were characterized by kinetic, chemical, and spectrometric methods, revealing conspicuous changes in their allosteric properties. The study of these mutants indicated that Arg172 which is located in the highly flexible motif 158-187 forming the active site lid has a specific role in binding the substrate to the enzyme in the T state. The possible role of this interaction in the conformational coupling of the active and the allosteric sites is discussed.
Anales de la Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
Die Naturwissenschaften, 1966
Nature, 1963
IT has been shown that in lenses of galactose-fed rats there is a decrease of glucose-6-phosphate... more IT has been shown that in lenses of galactose-fed rats there is a decrease of glucose-6-phosphate dehydrogenase1,2 and aldolase3. These findings support the statement that there is an interference of glucose metabolism in the lens during the development of galactose cataracts. In the previous experiments2 glutathione reductase was also determined as a coupled system with the glucose-6-phosphate dehydrogenase through the utilization of the oxidized and reduced forms of nicotinamide-adenine dinucleotide phosphate (NADP). The activity of the glutathione reductase was reduced similarly to the glucose-6-phosphate dehydrogenase. Incubation of normal lens homogenate with galactose or galactose-1-phosphate showed no inhibitory effect on the glucose-6-phosphate dehydrogenase or aldolase activities2,3. Considering these results, and also that a lowered amount of soluble proteins per mg lens was found in the lactose-fed rats3, we came to the conclusion that this was due to a decreased protein synthesis in lenses in this condition. However, aldolase was more affected than glucose 6-phosphate dehydrogenase, which may indicate a more direct action on the glycolytic pathway, which is, quantitatively, the most important pathway of glucose metabolism in lens4. The former experiments were performed independently with two different sets of rats. In order to know which of the two pathways is affected first, another group of animals was examined.
Experimental Parasitology, 1967
KORC, ISRAEL, HIERRO, Josh, LASALVIA, EDUARDO, FALCO, MARIO, AND CALCAGNO, MARIO. 1967. Chemical ... more KORC, ISRAEL, HIERRO, Josh, LASALVIA, EDUARDO, FALCO, MARIO, AND CALCAGNO, MARIO. 1967. Chemical characterization of the polysaccharide of the hydatid membrane of Echinococcus granulosus.
Experimental Cell Research, 1970
Action of cortisol and cortisone 2.5 x lo+ M on the metabolism of mucopolysaccharides (MPS) is st... more Action of cortisol and cortisone 2.5 x lo+ M on the metabolism of mucopolysaccharides (MPS) is studied in the femur of chick embryos cultivated in the usual medium. Under these experimental conditions, no alterations in DNA content are detected in cultures when treated with-cortisone. Untake of sulfate *?!I contained in the culture medium. measured in the CS isolated individuallv from cultures, is observed to increase with cortisol andcortisone treatment, which effect is intensified with prolonged administration. Similar results are obtained using "C glucose in experiments involving 4 days of treatment. The decay of CS labelled either with 9 or 14C follows marked narallel curves between treated cultures and controls and is not altered bv treatment. Autoradiography confirms the increase in sulphate incorporation and the determination of localization. The possible significance of these results, which differ from those described by various authors under slightly different experimental conditions, is discussed.
Revista De Ensino De Bioquimica, Jul 4, 2006
Although enzyme control and regulation is an important topic in most Biochemistry and Enzymology ... more Although enzyme control and regulation is an important topic in most Biochemistry and Enzymology courses, laboratory activities that allow an experimental approach to cooperativity and allosterism are difficult to implement. The objective of this work was to develop a simple and inexpensive experimental activity to teach this topic in basic courses. We decided to use the enzyme glucosamine-6-phosphate deaminase (GNPD, E.C. 3.5.99.6) from Escherichia coli, that is both kinetically and structurally well-known. GNPD is an allosteric enzyme, activated by Nacetylglucosamine 6-phosphate, that catalyzes the conversion of glucosamine 6-phosphate into fructose 6-phosphate and ammonia. The enzyme is a typical allosteric K-system and can be well described by the Monod-Wyman-Changeux (MWC) model. GNPD was partially purified through anionic-exchange chromatography from a mutant E.coli strain which expresses constitutively high levels of the enzyme. In order to measure activity we used an end point method which consists in stopping the reaction at a certain time point with HCl 10 N, and quantifying the fructose-6-phosphate formed with resorcinol (Selliwanoff reaction) through the formation of a red color that is measured spectrophotometrically. We developed a protocol that consisted in a 4-hour experiment in which the students measured the activity of the GNPD with increasing concentrations of the substrate, in the presence or absence of allosteric modulator. The students obtained a good quality data set that they analyzed based on the equations of Hill, MWC and Acerenza-Mirzaji (Biochim Biophys Acta, 1997Acta, , 1339. This protocol was used during the 2005 Enzymology course in Montevideo with very satisfactory results.
Journal of Bacteriology, 2016
We have investigated the impact of growth on glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) ... more We have investigated the impact of growth on glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) on cellular metabolism by quantifying glycolytic metabolites in Escherichia coli. Growth on GlcNAc increased intracellular pools of both GlcNAc6P and GlcN6P 10 to 20-fold, compared to growth on glucose. Growth on GlcN produced a 100-fold increase in GlcN6P but only a slight increase in GlcNAc6P. Changes to the amounts of downstream glycolytic intermediates were minor compared to growth on glucose. The enzyme glucosamine-6P deaminase (NagB) is required for growth on both GlcN and GlcNAc. It is an allosteric enzyme in E. coli, displaying sigmoid kinetics with respect to its substrate, GlcN6P and is allosterically activated by GlcNAc6P. The high concentration of GlcN6P, accompanied by the small increase in GlcNAc6P, drives NagBEc into its high activity state, as observed during growth on GlcN (Álvarez-Añorve et al. J Bacteriol 191:6401-7, 2009, doi: 10.1128/JB.00633-09). The slight increase in GlcNAc6P during growth on GlcN is insufficient to displace NagC, the GlcNAc6P-responsive repressor of the nag genes, from its binding sites, so there is only a small increase in nagB expression. We replaced the gene for the allosteric NagBEc enzyme with that of the non-allosteric, B. subtilis homologue, NagBBs.We detected no effects on growth rates or competitive fitness on glucose or the amino sugars, nor on the concentrations of central metabolites, thus demonstrating the robustness of amino sugar metabolism and leaving open the question of the role of allostery in the regulation of NagB. Chitin, the polymer of N-acetylglucosamine, is an abundant biomaterial and both glucosamine and N-acetylglucosamine are valuable nutrients for bacteria. The amino sugars are components of numerous essential macromolecules including bacterial peptidoglycan and mammalian glycosaminoglycans. Controlling the biosynthetic and degradative pathways of amino sugar metabolism is important in all organisms to avoid loss of nitrogen and energy via a futile cycle of synthesis and breakdown. The enzyme glucosamine-6P deaminase (NagB) is central to this control and N-acetylglucosamine-6P is the key signaling molecule regulating amino sugar utilization in E. coli. Here we investigate how the metabolic status of the bacteria impacts on the activity of NagBEc and the N-acetylglucosamine-6P-sensitive transcriptional repressor, NagC.
The intracellular concentration of the enzyme glucosamine-6-phosphate synthase, encoded by the ge... more The intracellular concentration of the enzyme glucosamine-6-phosphate synthase, encoded by the gene gimS in Escherichia coli, is repressed about threefold by growth on the amino sugars glucosamine and N-acetylglucosamine. This regulation occurs at the level of gimS transcription. It is not due just to the presence of intracellular amino sugar phosphates, because mutations which derepress the genes of the nag regulon (coding for proteins involved in the uptake and metabolism ofN-acetylglucosamine) also repress the expression ofglmS in the absence of exogenous amino sugars.
Journal of Molecular Biology, 2002
in the above paper was printed in error with the left and right-hand sides transposed. The correc... more in the above paper was printed in error with the left and right-hand sides transposed. The corrected part 'a' of the figure is reprinted here.
Biochimica Et Biophysica Acta Protein Structure and Molecular Enzymology, Jan 29, 1991
The secondary structure of the purified glucoseanine-6-phosphate deaminase from Eseherichia coli ... more The secondary structure of the purified glucoseanine-6-phosphate deaminase from Eseherichia coli KI2 was investigated by both circular dichro[sm (CD) spectroscopy and empirical prediction methods. The enzyme was obtained by allosteric-site affinity chromatography from an overproducing sWain bearing a pUCI8 plasmid carrying the structural gene for the enzyme. From CD analysis, 34% of a.helix, 9% of parallel ~-sheet, i1% of anfiparallel ~q-sheet, 15% turns and 35% of non-repetitive structures, were estimated. A joint prediction scheme, combining six prediction methods with defined rules using several physicochemical indices, gave the following values: a-helix, 37%; ~-sheet, 22%; turns, 18% and coil, 23%. The structure predicted showed also a considerable degree of alternacy of a and/] structures; 64% of helices are amphipathic and 90% of ~8-sheets are hydrophohic. Overall, the data suggest that deaminase has as dominaut motif, an a//3 sing'tore.
Journal of bacteriology, 1993
The intracellular concentration of the enzyme glucosamine-6-phosphate synthase, encoded by the ge... more The intracellular concentration of the enzyme glucosamine-6-phosphate synthase, encoded by the gene glmS in Escherichia coli, is repressed about threefold by growth on the amino sugars glucosamine and N-acetylglucosamine. This regulation occurs at the level of glmS transcription. It is not due just to the presence of intracellular amino sugar phosphates, because mutations which derepress the genes of the nag regulon (coding for proteins involved in the uptake and metabolism of N-acetylglucosamine) also repress the expression of glmS in the absence of exogenous amino sugars.
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Papers by Mario L. Calcagno