Peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3) catalyses the rate-limiting step in the post-translational activation of substance P, among other neuropeptides, from its glycine-extended precursor. Comparative kinetic studies were performed, using trans-styrylacetic acid or trans-styrylthioacetic acid as known mechanism-based inhibitors, of PHM isolated from rat, horse or human blood serum. Distinctive species differences with respect to PHM inactivation were observed: the efficiency of inactivation decreased in the order of horse >> rat > human. Trans-styrylacetic acid was more active than its thioether derivative. Moreover, we studied the differential sensitivity towards mechanism-based inactivation, of soluble PHM from rat blood serum and rat brain by trans-styrylacetic acid or benzylhydrazine, as well as the membrane-associated enzymes from rat brain and heart atrium. For the heart atrium membrane PHM or the soluble PHM from blood serum, inactivation rate constants k(inact)/K(I) of approximately 100 M(-1)sec(-1) were found with trans-styrylacetic acid. However, neither of the two tested compounds, at 100 microM or 12 mM, respectively, could inactivate the soluble or membranous PHMs from rat brain during a 15-min pre-incubation period. Instead, under conditions of reversible inhibition, trans-styrylacetic acid competitively inhibited the soluble or membrane-associated brain PHM with inhibition constants K(I) = 0.6 microM and 1.0 microM, respectively. Organ-selective, time-dependent inactivation of PHM with compounds of the above types might be an important pharmacological tool to control peripheral neuropeptide activation.