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.