Inactivation kinetics of the reduced spinach chloroplast fructose-1,6-bisphosphatase by subtilisin.
The course of inactivation of the reduced spinach chloroplast fructose-1,6-bisphosphatase by digestion with subtilisin has been followed by the progress curve method [Tsou, C. L. (1988) Adv. Enzymol. 61, 381-436] and found to follow first-order kinetics. On the basis of the hydrolysis of the substrate, fructose 1,6-bisphosphate, at different concentrations during proteolysis by subtilisin, the first-order inactivation rate constants for the free enzyme and the enzyme-substrate complex can both be determined. The ratio between the inactivation rate constants for the free enzyme and the enzyme-substrate complex indicates strong protection against subtilisin proteolysis by the substrate. It is proposed that the above ratio can be used as a quantitative measure of substrate protection for enzyme inactivation generally. As it has been found that the site of proteolysis is located in a loop region near the N-terminus and well away from the active site, the substrate protection indicates a conformation change of the enzyme away from the substrate binding site.
Chen
Y
Y
National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Bejiing, China.
Wu
J W
JW
Xu
G J
GJ
Tsou
C L
CL
Wang
Z X
ZX
D001665:Binding Sites; D002736:Chloroplasts / Q000201:enzymology*; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006597:Fructose-Bisphosphatase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D011487:Protein Conformation; D018724:Spinacia oleracea / Q000201:enzymology*; D013381:Subtilisins / Q000378:metabolism / Q000494:pharmacology*
D004220:Disulfides; D006597:Fructose-Bisphosphatase; D013381:Subtilisins; D004229:Dithiothreitol