Inhibition studies in situ of yeast catalases.

The catalase activity of the intact yeast cells towards external substrate is generally lower than the 'cryptic' activity which is revealed after cell lysis. The physiological basis for the reduced catalytic activity of the intact cell ('patent' activity) has been investigated by establishing the inhibition profiles of catalases in situ using selected probes; to this end we utilized either non-penetrating acids and/or catalase poisons able to cross the plasmic membrane. Owing to the peculiar features of the reaction mechanism, competitive inhibitors, which are known to interact with the prosthetic group of catalases, show an efficiency that is unlinked to the hydrogen peroxide concentration under the usual assay conditions ([H2O2] much less than Km). This mode of interaction, which also characterizes the action of the penetrating probes HCOOH and HCN, is particularly well adapted to the study of the behaviour of the cytoplasmic catalases in situ. By this experimental approach, it has been shown that the catalase of the inner cellular region contributes, together with an isoenzyme present at the cell surface, to the patent activity. The mathematical processing of the data, which takes into account a rate-limiting diffusion of external substrate into the intact yeast cell, has allowed us to predict accurately the resulting apparent efficiency of inhibitors as a function of the physiological variations of the intracellular enzyme concentration.