A kinetic study of the inhibition of palmito polyphenol oxidase by L-cysteine

Polyphenol oxidase (PPO) is responsible for browning reactions in fruits and vegetables. In order to inhibit these reactions during processing, the use of chemical inhibitors including thiols such as L-cysteine has been proposed. The effect of this thiol on PPO activity was studied in order to establish if it reacts with quinone and/or directly inhibits the enzyme. The inhibition of palmito PPO (catecholase activity, with 4-methylcatechol as substrate) by L-cysteine was characterized spectrophotometrically. The effect of increasing cysteine concentration with respect to incubation time was measured by polarography and the kinetic parameters calculated. Kinetic analysis using spectrophotometric assays showed a lag period suggesting that there is no quinone accumulation in the reaction mixture. The increase of absorbance at 300 nm observed with u.v.-vis spectra indicated that a colourless compound (thiol-quinone complex) was produced. The plots of log remaining enzyme activity vs incubation time were characterized by two straight lines suggesting two possible inactivation models: (i) the deactivation of one enzyme that proceeds in two steps or (ii) the existence of two isoenzymes that behave differently. We have shown that the inactivation process of PPO by cysteine followed this model: N --> X\C\ --> I where N represents one native form, X represents an intermediate form, the structure of which depends on the cysteine concentration [C], and where I is the completely inactive form of the enzyme. These results showed that there is a direct irreversible inhibition of PPO by cysteine according to a two-step model. The analytical approach illustrated by the present study of palmito PPO inhibition by L-cysteine may be applied to other investigations of mechanisms of enzyme inhibition. Copyright (C) 1996 Elsevier Science Ltd.