Kinetics of the slow pH-mediated transition of polyphenol oxidase

Catecholase activity of latent polyphenol oxidase from broad bean leaves showed a hysteresis phenomenon above pH 4, whereas a steady-state rate was reached immediately when pH values were lower, thus suggesting that slow pH-induced conformational changes in the protein occur during the assay. When the enzyme was activated by sodium dodecyl sulfate, the lag period completely disappeared. This transition was reversible, since a burst was observed when the enzyme was preincubated at acid pH, before being returned to its previous experimental conditions. The pK for the isomerization process (pK(H) = 4.6) was estimated by preincubating the enzyme at different pH values and analyzing the product accumulation curves. Negative kinetic cooperativity was evident over a pH range in which the isomerization reaction was significant when the steady state was measured as a function of different substrate concentrations.