Chemical modification studies of the active site of glucosamine-6-phosphate synthase from baker's yeast

• Published in: Biochimica et biophysica acta Vol. 1161; no. 2; pp. 279 - 284
• Main Author: Milewski, Sławomir
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 13.02.1993; Elsevier
• Subjects: Analytical, structural and metabolic biochemistry; Baker's yeast; Binding Sites; Biological and medical sciences; Chemical modification; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Glucosamine-6-phosphate synthase; Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) - metabolism; Irreversible modification; Kinetics; Molecular Structure; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Transferases; DPC, diethylpyrocarbonate; FMDP, N 3-(4-methoxyfumaroyl)- l-2,3-diaminopropanoic acid; Irreversible modification; CMC, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide-metho-4-toluene sulfonate; NTCB, 2-thiocyano-5-nitrobenzoic acid; Hepes, N-(2- hydroxyethyl) piperazine-N′-(2- ethanesulfonic acid); 2,3-BD, 2,3-butanedione; Mes, 2-(N- morpholino)ethanesulfonic acid; DON, 6-diazo-5-oxo- l-norleucine; Glucosamine-6-phosphate synthase; GlcN-6-P, d-glucosamine 6-phosphate; Chemical modification; PMSF, phenylmethylsulfonyl fluoride; PLP, pyridoxal 5′-phosphate; Baker's yeast; FCDP, N 3-fumaramoyl- l-2,3-diaminopropanoic acid; d-Fru-6-P, d-Fructose 6-phosphate; Yeast; Purification; Enzyme; Active site; Enzyme inhibitor; Fungi; Affinity chromatography; Glucosaminephosphate isomerase (glutamine-forming); Ascomycetes; Kinetic parameter; Saccharomyces cerevisiae; Thallophyta
• ISSN: 0167-4838; 0006-3002; 1879-2588
• DOI: 10.1016/0167-4838(93)90225-G

Glucosamine-6-phosphate synthase from baker's yeast has been purified 100-fold with a final recovery of 70%. The purification procedure involved thiol-affinity chromatography. Chemical modification studies of the enzyme revealed the presence of cysteine, Glu/Asp-carboxyl and probably histidine at the glutamine binding site and, on the other hand, arginine and probably another histidine at the d-fructose 6-phosphate binding site. A few glutamine analogs, including 6-diazo-5-oxo- l-norleucine (DON), anticapsin and N 3-(4-methoxyfumaroyl)- l-2,3-diaminopropanoic acid (FMDP), were shown to inactivate the enzyme in a time- and concentration-dependent manner. Anticapsin, the most active in the series, exhibited an inactivation constant, K inact , of 9.5 · 10 −6 M.