3 , 4 = Dioxygenase III

In the early stage of the reaction of protocatechuate 3,4dioxygenase, a new spectral species of the enzyme having a broad absorption band with a maximum between 500 and 520 nm was observed by using a stopped flow technique. The observed spectrum was distinct from those of the enzyme or the enzyme-protoacatechuic acid complex and its formation was absolutely dependent on the simultaneous presence of both oxygen and substrate, protoacatechuic acid. The rate constant for the decomposition of the new spectral species determined by stopped flow experiments was in good agreement with the turnover number of the enzyme determined from the over-all reaction. A similar spectral species was also observed during the steady state of the reaction when enzymatically active substrate analogues such as 3,4-dihydroxyphenylacetic acid or 3,4-dihydroxyphenylpropionic acid were used. The rate constants for the decomposition of the new spectral species were also in accord with their turnover numbers of the enzyme. Further analysis of the detailed kinetics with these substrate analogues revealed that the rates of the over-all reaction at different time intervals were proportional to the amount of the intermediate present and the integrated amounts of the intermediate during the entire course of the reaction were also proportional to the initial concentrations of oxygen. These results indicate that the new spectral species is a ternary complex of oxygen, substrate, and enzyme, and its degradation is the rate-limiting step of the over-all reaction. The rate constants for the formation of the enzyme-substrate complex and of the ternary complex were also determined with substrate analogues. Kinetic parameters calculated from rate constants were also in good agreement with those obtained from the over-all reaction.