Biosynthesis of bacterial glycogen. Use of site-directed mutagenesis to probe the role of tyrosine 114 in the catalytic mechanism of ADP-glucose synthetase from Escherichia coli.
Previous covalent modification studies showed that tyrosine 114 of Escherichia coli ADP-glucose synthetase is involved in substrate binding (Lee, Y. M., and Preiss, J. (1986) J. Biol. Chem. 261, 1058-1064). We have prepared, via site-directed mutagenesis, an E. coli ADP-glucose synthetase variant (Phe114) containing a Tyr114 to Phe substitution in order to test whether the phenolic hydroxyl group plays a critical role in catalysis. Kinetic characterization of Phe114 ADP-glucose synthetase indicates that the Tyr114 hydroxyl is not obligatory for the enzyme catalysis. However, the variant enzyme showed altered properties. It showed a decreased apparent affinity for the substrates. The variant enzyme showed less than 2-fold activation by 5 mM fructose 1,6-bisphosphate in the ADP-glucose synthesis direction. In contrast, in the pyrophosphorolysis direction, the mutant enzyme showed about a 30-fold activation by 5 mM fructose 1,6-bisphosphate. The variant enzyme is heat-labile compared to wild type enzyme. It lost about 60% enzyme activity on incubation at 65 degrees C for 5 min in the presence of 30 mM Pi. The wild type enzyme is stable under these conditions. The results indicate that tyrosine 114 is involved directly or indirectly in enzyme catalysis, but is not obligatory for the enzyme catalysis. Conversion of Tyr114 to Phe also alters the regulatory properties of the enzyme with respect to activation by fructose-1,6-P2 and inhibition by AMP.
Kumar
A
A
Department of Biochemistry, Michigan State University, East Lansing 48824.
Tanaka
T
T
Lee
Y M
YM
Preiss
J
J
D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D051599:Glucose-1-Phosphate Adenylyltransferase; D006003:Glycogen / Q000096:biosynthesis*; D006358:Hot Temperature; D007700:Kinetics; D009154:Mutation; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine
D014443:Tyrosine; D006003:Glycogen; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase