DIVERSE EFFECTS OF PH ON THE INHIBITION OF HUMAN CHYMASE BY SERPINS

Inhibition of human chymase by the serpins alpha(1)-antichymotrypsin ( ACT) and alpha(1)-proteinase inhibitor (PI) at pH 8.0 produces a compl ex stable to dissociation by SDS/dithiothreitol and a second product, hydrolyzed/inactivated serpin, The first product is the presumed trapp ed acyl-enzyme complex typical of serpin inhibition, and the second is the result of a concurrent substrate-like reaction, As a result of th e hydrolytic reaction, stoichiometries of inhibition (SI) appear great er than 1; values of 4 and 6.0 are observed for the chymase-ACT and -P I reactions, In this study the effect of pH on the inhibition rate con stant (k(inh)) and the SI of each reaction were evaluated to better de fine the rate-limiting steps of the inhibitory and hydrolytic reaction pathways associated with chymase inhibition, Reactions were evaluated over a pH range to correlate k(inh) and SI with the ionizations (pK v alues of 7 and 9) that typically regulate serine protease catalytic ac tivity, The results show that the effects of pH on SI and k(?inh), dif fer for each inhibitor, On reducing the pH from 8.0 to 5.5, the chymas e-ACT reaction exhibited a decrease in SI (to about 1) and little chan ge in k(inh) whereas the chymase PI reaction revealed an increase in S I and a marked decrease in k(inh). On increasing the pH from 8.0 to 10 .0, the chymase-ACT reaction exhibited little change in SI and a marke d decrease in k(inh) whereas the chymase-PI reaction revealed a decrea se in SI and a marked increase in k(inh). Chymase catalytic properties determined for a peptide substrate were atypical over the high pH ran ge exhibiting increases for k(cat)/K-m and k(cat) and decreases for K- m. This behavior suggests the presence of a high pH enzyme form with e nhanced hydrolytic activity. From these results and others involving a nalyses of ACT/PI reactive loop chimeras and ACT point variants exhibi ting a range of SI values, we suggest that the diverse pH effects on k (inh) and SI are caused largely by a difference in the abilities of AC T and PI to interact with low (catalytically inactive) and high (catal ytically enhanced) pH forms of chymase, The constancy of k(inh) for th e chymase-ACT reaction over the low pH range suggests that the rate-li miting step for inhibition is pH insensitive and not reflective of dim inished chymase hydrolytic activity, Low pH did not appear to affect t he rate of SDS-stable complex formation as complex accumulation, asses sed qualitatively by SDS-PAGE, correlated with the loss of chymase enz ymatic activity.