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.
Altamirano
M M
MM
Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City, D.F.
Mulliert
G
G
Calcagno
M
M
D000494:Allosteric Regulation; D055598:Chemical Phenomena; D002621:Chemistry; D003545:Cysteine / Q000032:analysis; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation; D004793:Enzyme Reactivators; D004926:Escherichia coli / Q000201:enzymology*; D005956:Glucose-6-Phosphate Isomerase / Q000032:analysis / Q000378:metabolism*; D006847:Hydrocarbons, Iodinated / Q000494:pharmacology; D007700:Kinetics; D010504:Periodic Acid / Q000494:pharmacology; D011489:Protein Denaturation; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000032:analysis; D014508:Urea
D004793:Enzyme Reactivators; D006847:Hydrocarbons, Iodinated; D013438:Sulfhydryl Compounds; D010504:Periodic Acid; D014508:Urea; D004228:Dithionitrobenzoic Acid; C009288:metaperiodate; C014055:methyl iodide; D005956:Glucose-6-Phosphate Isomerase; D003545:Cysteine; D004229:Dithiothreitol