Differential scanning calorimetric study of 5-enolpyruvoyl shikimate-3-phosphate synthase and its complexes with shikimate-3-phosphate and glyphosate: irreversible thermal transitions.
The thermal denaturation of native Escherichia coli 5-enolpyruvoyl shikimate-3-phosphate (EPSP) synthase, its binary complex with shikimate-3-phosphate (S3P) and its ternary complex with S3P and glyphosate have been studied using highly-sensitive differential scanning calorimetry (DSC). All observed transitions are strongly scanning-rate-dependent and irreversible. Consistent with these observations, the data were better fit by a simple irreversible model than by the controversial reversible model more commonly employed. The results obtained provide additional support for the application of irreversible models to the thermal denaturation of proteins. The calculated parameters, activation energy (Ea), enthalpy of denaturation (delta H) and transition temperature (Tm), obtained from fitting to an irreversible model agree well with values obtained from approximation techniques. Further, the results show that the formation of the ternary complex greatly enhances the thermal stability of the enzyme (delta Tm = 10.6 degrees C), while the binding of S3P alone increases the transition temperature only slightly (delta Tm = 3 degrees C). The heat of binding calculated at the transition temperature also demonstrates the greater stability of the ternary complex (delta H = -70 kcal/mol) versus the binary complex (delta H = -10 kcal/mol).
Merabet
E K
EK
New Products Division, Monsanto Agricultural Co., St. Louis, MO.
Walker
M C
MC
Yuen
H K
HK
Sikorski
J A
JA
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D002152:Calorimetry, Differential Scanning; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D008962:Models, Theoretical; D011489:Protein Denaturation; D012765:Shikimic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D013816:Thermodynamics; D014166:Transferases / Q000737:chemistry* / Q000378:metabolism
D012765:Shikimic Acid; C042786:shikimic acid-3-phosphate; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase