D-glucosaminate aldolase activity of D-glucosaminate dehydratase from Pseudomonas fluorescens and its requirement for Mn2+ ion.
When D-glucosaminate dehydratase (GADH) was incubated with D-glucosaminate (GlcNA) in veronal buffer (VB; 0.01 M, pH 8.0), GlcNA was converted stoichiometrically to glyceraldehyde, pyruvate, and ammonia (aldolase reaction A). This reaction occurred in addition to the dehydratase reaction (conversion of GlcNA to 2-keto-3-deoxy-D-gluconate and ammonia: alpha,beta-elimination reaction, B). The ratio of the activities (A:B) was about 1:4. However, in potassium phosphate buffer (KPB; 0.04 M, pH 8.0), the aldolase reaction was inhibited to 3-4% of that in VB, and also inhibited by various derivatives of glycerol, in particular, glycerol-3-phosphate (glycerol-3-P) and glyceraldehyde-3-phosphate (glyceraldehyde-3-P) in VB. The native enzyme was inhibited by incubation with 0.1 M EDTA, and the activity was restored by incubation of the EDTA-treated enzyme with (Mn2+ + pyridoxal 5'-phosphate (PLP)). When the EDTA-treated enzyme was incubated with (Mn2+ + PLP + glycerol-3-P), the activity of reaction B increased to 131% but that of reaction A decreased to 21%. These results suggested that Mn2+, PLP, and the phosphate group of glycerol-3-P are involved in formation of the active enzyme. In the case of the aldolase reaction, Mn2+ ion, which might be essential for the reaction, is chelated by the phosphate group of glycerol-3-P with resultant inhibition of the aldolase reaction.
Iwamoto
R
R
Department of Chemistry, Faculty of Science, Nara Women's University, Japan.
Taniki
H
H
Koishi
J
J
Nakura
S
S
D000446:Aldehyde-Lyases / Q000302:isolation & purification / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D004492:Edetic Acid / Q000494:pharmacology; D005990:Glycerol / Q000494:pharmacology; D006836:Hydro-Lyases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008345:Manganese / Q000378:metabolism*; D011551:Pseudomonas fluorescens / Q000201:enzymology*
D008345:Manganese; D004492:Edetic Acid; D000446:Aldehyde-Lyases; D006836:Hydro-Lyases; C018870:aminodeoxygluconate dehydratase; D005990:Glycerol