Modification of uridine phosphorylase from Escherichia coli by diethyl pyrocarbonate. Evidence for a histidine residue in the active site of the enzyme

Uridine phosphorylase from Escherichia coli is inactivated by diethyl pyrocarbonate at pH 7.1 and 10 degrees C with a second-order rate constant of 840 M-1.min-1. The rate of inactivation increases with pH, suggesting participation of an amino acid residue with pK 6.6. Hydroxylamine added to the ina...

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Bibliographic Details
Published in:Biochemical journal 1990-09, Vol.270 (2), p.319-323
Main Authors: Drabikowska, A K, Woźniak, G
Format: Article
Language:English
Subjects:
Binding Sites
Diethyl Pyrocarbonate - pharmacology
Escherichia coli
Escherichia coli - enzymology
Formates - pharmacology
Histidine
Hydrogen-Ion Concentration
Kinetics
Pentosyltransferases - antagonists & inhibitors
Uridine - pharmacology
Uridine Phosphorylase - antagonists & inhibitors
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Summary:Uridine phosphorylase from Escherichia coli is inactivated by diethyl pyrocarbonate at pH 7.1 and 10 degrees C with a second-order rate constant of 840 M-1.min-1. The rate of inactivation increases with pH, suggesting participation of an amino acid residue with pK 6.6. Hydroxylamine added to the inactivated enzyme restores the activity. Three histidine residues per enzyme subunit are modified by diethyl pyrocarbonate. Kinetic and statistical analyses of the residual enzymic activity, as well as the number of modified histidine residues, indicate that, among the three modifiable residues, only one is essential for enzyme activity. The reactivity of this histidine residue exceeded 10-fold the reactivity of the other two residues. Uridine, though at high concentration, protects the enzyme against inactivation and the very reactive histidine residue against modification. Thus it may be concluded that uridine phosphorylase contains only one histidine residue in each of its six subunits that is essential for enzyme activity.
ISSN:0264-6021
1470-8728
DOI:10.1042/bj2700319