Abstract
D-Glucosaminate dehydratase (EC 4.2.1.26) from Pseudomonas fluorescens (IFO 14808) was purified to homogeneity, as judged by the criterion of a single band on disc-gel electrophoresis. The enzyme (molecular weight 61, 000) consisted of two subunits identical in molecular weight (about 30, 000). Pyridoxal 5'-phosphate was an essential cofactor for the enzyme.
The enzyme catalyzed the dehydration of D-glucosaminate and of several D-and L-hydroxyamino acids. Especially when the concentration of substrate was low, D-serine was dehydrated at a rate similar to the rate of dehydration of D-glucosaminate. If both substrates were added to the reaction mixture at the same time, the rate of the reaction was additive until their individual concentrations reached to 0.2 mM level. However, as the concentrations of both the substrates were increased, the rate fell below the rate recorded with D-glucosaminate only. The kinetics of this reaction (in the presence of 1-10mM D-GlcNA) demonstrate that D-glucosaminate dehydratase is inhibited both noncompetitively (at low concentrations) and competitively by D-serine. From these and other observations, we postulate the presence of a binding site specific for D-serine on the enzyme.
