Mechanistic studies of glutamine synthetase from Escherichia coli: kinetics of ADP and orthophosphate binding to the unadenylylated enzyme

Biochemistry
S G Rhee, P B Chock

Abstract

The kinetics of protein fluorescence change exhibited by ADP or orthophosphate addition to the Mg2+-or Mn2+-activated unadenylylated glutamine synthetase from Escherichia coli were studied. The kinetic patterns of these reactions are incompatible with a simple bimolecular binding process and a mechanism which required protein isomerization prior to substrate binding. They are consistent with a mechanism in which direct substrate binding is followed by a substrate-induced conformational change step, ES in equilibrium ES. At pH 7.0 and 15 degrees C, the association constants for the direct binding (K1) of ADP to MnE1.0 and of Pi to MnE1.0ADP are 3.9 X 10(4) and 2.28 X 10(2) M(-1), respectively. The association constant for the direct binding of ADP to MnE1.0Pi is 2.3 X 10(4) M(-1) at pH 7.0 and 19 degrees C. The deltaG degrees for the substrate-induced conformational step are -3.5 and -1.3 kcal mol(-1) due to ADP binding to MnE1.0Pi and MnE1.0, respectively, and -1.4 kcal mol(-1) due to Pi binding to MnE1.0ADP. Rate constants, k2 and k(-2), for the isomerization step are: 90 and 9.5 s(-1) for ADP binding to MnE1.0, 440 and 0.36 s(-1) for ADP binding to MnE1.0Pi, and 216 and 1.8 s(-1) for Pi binding to MnE1.0ADP. Due to low subst...Continue Reading

References

Feb 1, 1976·Proceedings of the National Academy of Sciences of the United States of America·S G Rhee, P B Chock
Jan 1, 1975·Archives of Biochemistry and Biophysics·J B HuntE R Stadtman
Sep 1, 1975·Proceedings of the National Academy of Sciences of the United States of America·S ShaltielE R Stadtman
Aug 1, 1967·Proceedings of the National Academy of Sciences of the United States of America·B ShapiroE R Stadtman
Sep 26, 1966·Archives of Biochemistry and Biophysics·C A WoolfolkE R Stadtman

Citations

Jan 1, 1979·The International Journal of Biochemistry·J J Villafranca, M S Balakrishnan
Jun 1, 1993·Proceedings of the National Academy of Sciences of the United States of America·S H LiawD Eisenberg
Mar 21, 1977·Biochemical and Biophysical Research Communications·J J VillafrancaF C Wedler
Mar 30, 2001·The Journal of Biological Chemistry·L BrochierD Fournier

Related Concepts

Magnesium ADP
Enzyme Activation
Alkalescens-Dispar Group
Glutamate-Ammonia Ligase
Magnesium
Manganese
Mathematics
Orthophosphate
Plasma Protein Binding Capacity
Fluorescence Spectroscopy

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