Inactivators in competition -: How to deal with them ... and not!

A method is described to determine the values of the equilibrium (K) and rate (k(2)) constants for enzyme inactivations which occur according to two-step pathways involving a first non-covalent complex and a covalent, irreversibly inactivated adduct. The method rests on a competition between a reference compound [R] for which the k(2) and K values are already known and another inactivator [C]. During the experiments, the disappearance of the reference compound or the appearance of the ER* adduct is monitored. The analysis shows that under conditions where the k(2) and K values for the competing substrate can be determined, the measured apparent first-order rate constant for the disappearance of the reference compound is not the sum of the rate constants obtained for each inactivator in the absence of the other. The method can be used to determine the K and k(2) constants when an adequate reference compound is available, in particular, for the interactions between beta-lactam antibiotics and penicillin-binding proteins. The precautions which must be taken to avoid large errors on the estimation of the parameters of the competing inactivator are discussed. Examples found in the literature are discussed where an erroneous simplified equation has been utilised, thus yielding incorrect values for k(2) and K. Interestingly, the correct values can be calculated on the basis of the published results which do not contain the raw experimental data. But some of the values should be considered with a lot of caution since the experiments have not been performed under optimal conditions. (c) 2005 Elsevier Inc. All rights reserved.