Biomedical subjects

M M Altamirano

Publications and source records attributed to M M Altamirano.

22 records · Page 2Linked to original sources

PubMed1991

Secondary structure of Escherichia coli glucosamine-6-phosphate deaminase from amino acid sequence and circular dichroism spectroscopy.

The secondary structure of the purified glucosamine-6-phosphate deaminase from Escherichia coli K12 was investigated by both circular dichroism (CD) spectroscopy and empirical prediction methods. The enzyme was obtained by allosteric-site affinity chromatography from an overproducing strain bearing a pUC18 plasmid carrying the structural gene for the enzyme. From CD analysis, 34% of alpha-helix, 9% of parallel beta-sheet, 11% of antiparallel beta-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: alpha-helix, 37%; beta-sheet, 22%; turns, 18% and coil, 23%. The structure predicted showed also a considerable degree of alternacy of alpha and beta structures; 64% of helices are amphipathic and 90% of beta-sheets are hydrophobic. Overall, the data suggest that deaminase has as dominant motif, an alpha/beta structure.

Aldose-Ketose Isomerases↗

PubMed1990

Zinc binding and its trapping by allosteric transition in glucosamine-6-phosphate deaminase from Escherichia coli.

Glucosamine-6-phosphate isomerase deaminase from Escherichia coli, a typical allosteric enzyme, becomes less cooperative and 50% inhibited when treated with zinc. This metal cation behaving as a tight-bound and slow partial inhibitor. Modification of a pair of vicinal reactive thiols with some sulfhydryl reagents mimics this effect. On the other hand, sulfhydryl reactivity disappears in the presence of saturating concentrations of Zn2+, which does not modify the kinetics of S-methylated enzyme, a finding that indicates that vicinal thiols are an essential part of the zinc-binding site. Allosteric activation of the deaminase causes trapping of the metal, which cannot be released by dialysis against a buffer containing EDTA. Cadmium and nickel(II) cations also produce a similar effect.

Aldose-Ketose Isomerases↗

PubMed1989

Evidence for vicinal thiols and their functional role in glucosamine-6-phosphate deaminase from Escherichia coli.

Methylation of glucosamine-6-phosphate isomerase deaminase (2-amino-2-deoxy-D-glucose-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 Vmax/Etotal 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 (1,10-phenanthroline)3-Cu(II) 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.

Aldose-Ketose Isomerases↗

PubMed1987

Sulfhydryl groups of glucosamine-6-phosphate isomerase deaminase from Escherichia coli.

Glucosamine-6-phosphate isomerase deaminase (2-amino-2-deoxy-D-glucose-6-phosphate ketol isomerase (deaminating), EC 5.3.1.10) from Escherichia coli is an hexameric homopolymer that contains five half-cystines per chain. The reaction of the native enzyme with 5',5'-dithiobis-(2-nitrobenzoate) or methyl iodide revealed two reactive SH groups per subunit, whereas a third one reacted only in the presence of denaturants. Two more sulfhydryls appeared when denatured enzyme was treated with dithiothreitol, suggesting the presence of one disulfide bridge per chain. The enzyme having the exposed and reactive SH groups blocked with 5'-thio-2-nitrobenzoate groups was inactive, but the corresponding alkylated derivative was active and retained its homotropic cooperativity toward the substrate, D-glucosamine 6-phosphate, and the allosteric activation by N-acetyl-D-glucosamine 6-phosphate. Studies of SH reactivity in the presence of enzyme ligands showed that a change in the availability of these groups accompanies the allosteric conformational transition. The results obtained show that sulfhydryls are not essential for catalysis or allosteric behavior of glucosamine-6-phosphate deaminase.

Allosteric Regulation↗