Differential reactivity of chloroplast fructose-1,6-bisphosphatase to Woodward's reagent K and diethylpyrocarbonate

PRAT GAY G. DE; WOLOSIUK, R. A.

BIOCHIMICA AND BIOPHYSICA ACTA

Año: 1987 vol. 973 p. 457 - 464

0006-3002

Native chloroplast fructose-1,6-bisphosphatase (d-fructose 1,6-bisphosphate-l-phosphohydrolase, EC 3.1.3.11) wasinactivated with either Woodward´s reagent K or diethylpyrocarbonate. Since pseudo-first-order rate constants (κapp) were a hyperbolic function of inhibitor concentration, it appeared that native chloroplast fructose-1,6-bisphosphatase and either Woodward´s reagent K or diethylpyrocarbonate interacted reversibly prior to formation of irreversible (inactive) complex. Protection against inactivation was afforded by preincubating the enzyme with dithiothreitol, fructose 1,6-bisphosphate, Ca2+ and either chloroplast thioredoxin-f or a chaotropic anion (trichloroacetate). Following similar incubation with an organic solvent (2-propanol), the catalytic activity remained unaltered in the presence of Woodward´s reagent K but was inactivated by diethylpyrocarbonate. The enhancement of the specific activity of chloroplast fructose-1,6-bisphosphatase caused by dithiothreitol, fructose-1,6-bisphosphate, Ca2+ and either chloroplast thioredoxin-f, or chaotropic anions or cosolvents was similar. Therefore, differential reactivity to selected reagents indicated the existence of several conformations of chloroplast fructose-1,6-bisphosphatase, i.e., native and different active forms.