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The influence of the hydrogen ion concentration on catalytic constants (kcat, KM and kcat/KM) was investigated under steady-state conditions where all steps of the Scheme, are taken into the consideration. The relative value of the rate constants has an influence on pH dependence of catalytic constants. The pH of intersection points of the tangents at the portion of functions pKM, Ig kcat, lg kcat/KM = f(pH) with different slopes are the values of true pKa of catalytically important ionizable groups in the free enzyme or in the enzyme-substrate complex(es), when the conditions k'-1, k'1, k'2 = 0 are valid. If e.g. k'-1, k'1 not equal to 0 the pH of the intersection points of the tangents is sometimes not the true value of the pKa. In this connection it is possible that more than one intersection point exists (in the plot lg kcat, pKM or lg kcat/KM versus pH) when only one ionizing group in the active site of an enzyme is present.

Using Gly-Pro-pNa as a substrate, the authors photometrically determined the dipeptidyl peptidase activity in organic homogenates and serum of Wistar rats at two pH values. The activity of the serine peptidase DP IV was measured at pH = 7.6; a mixed activity (DP MA) which is composed of the activities of several dipeptidyl peptidases, at pH = 5.0. Particularly high DP IV activities were determined in the kidney, lung, adrenal gland, liver and aortic arch, the differences among the enzyme activities measured in the single organic homogenates at pH = 5.0 being smaller. The DP IV activities and the values found for DP MA in the structures of the nervous system were in general lower. Maximum DP IV values were measured in the spinal ganglion, cerebral cortex and spinal bulb; and maximum DP MA values, in the hypothalamus, cerebellum and cerebral cortex. The results obtained are discussed from the aspects of the participation of these enzymes in peptidergic regulatory mechanisms and of a possible relation to blood-pressure regulation.

Di-isopropyl fluorophosphate (DFP) and other organophosphorus inhibitors recruit the catalytic power of their target enzymes: the enzyme catalyzes its own irreversible phosphorylation. The magnitude of the catalytic acceleration can approach the factor by which the enzyme catalyzes its own acylation by natural substrates. The reaction of DFP with five serine proteases [chymotrypsin, elastase, dipeptidyl peptidase IV (DP IV), subtilisin and thermitase] exhibits in all cases the same pH dependence as does enzyme acylation by natural substrates. Chymotrypsin and elastase form a "fast" class of enzymes which react about ten-fold faster than the other three "slow" enzymes. All enzymes show k(HOH)/k(DOD) of about 2 but the proton inventory indicates one-proton character for "slow" enzymes and multiproton character for "fast" enzymes. Enthalpies of activation are about 33 kJ/mol (subtilisin, "slow") and 10 kJ/mol (elastase, "fast"). Entropies of activation are about -120 J.T-1.mol-1 (subtilisin, "slow") and -175J.T-1.s-1 (elastase, "fast"; T = temperature in K).

According to the increasing importance of enzyme catalysed peptide synthesis the capability of the protease dipeptidyl peptidase IV is shown to form the peptide bond. To our best knowledge, it's the first time that has been used successfully proline as one of the amino acids. The formation of the peptide bond is kinetically controlled. Free amino acids have been used as nucleophiles. The reaction media contain 50 vol.-% glycerol. A first approach to a kinetic treatment is shown.