APPLICATION OF IODINE-LASER TEMPERATURE-JUMP TO AN IRREVERSIBLE ENZYME REACTION - REACTION OF ASCORBIC-ACID WITH HYDROGEN-PEROXIDE CATALYZED BY PEROXIDASE

The problem of the determination of the rate of a fast intermediate step in an irreversible enzyme reaction which is not detectable by steady-state kinetics has been addressed. Peroxidase enzymes characteristically have two oxidized intermediate species formed in their reaction cycle, compounds I and II, that can both react with the same reducing substrate in one-electron reactions. Compound I typically reacts an order of magnitude faster than compound II and the steady-state concentration of compound I is therefore very small. By application of the iodine-laser temperature-jump relaxation method combined with fast mixing to the peroxide-catalysed reaction of hydrogen peroxide with ascorbic acid, we demonstrate, for the first time, that the rate of reduction of compound I can be determined directly while the reaction cycle is in progress.