Reactive-site specificity of human kallistatin toward tissue kallikrein probed by site-directed mutagenesis

Kallistatin is a serine proteinase inhibitor that forms complexes with tiss ue kallikrein and inhibits its activity. In this study, we compared the inh ibitory activity of recombinant human kallistatin and two mutants, Phe388Ar g (P1) and Phe387Gly (P2), toward human tissue kallikrein. Recombinant kall istatins were expressed in Escherichia coli and purified to apparent homoge neity using metal-affinity and heparin-affinity chromatography. The complex es formed between recombinant kallistatins and tissue kallikrein were stabl e for at least 150 h. Wild-type kallistatin as well as both Phe388Arg and P he387Gly mutants act as inhibitors and substrates to tissue kallikrein as a nalyzed by complex formation. Kinetic analyses showed that the inhibitory a ctivity of Phe388Arg variant toward tissue kallikrein is two-fold higher th an that of wild type (P1Phe), whereas Phe387Gly had only 7% of the inhibito ry activity toward tissue kallikrein as compared to wild type. The Phe388Ar g variant but not wild type inhibited plasma kallikrein's activity. These r esults indicate that P1Arg variant exhibits more potent inhibitory activity toward tissue kallikrein while wild type (P1Phe) is a more selective inhib itor of tissue kallikrein. The P2 phenylalanine is essential for retaining the hydrophobic environment for the interaction of kallistatin and kallikre in. (C) 2000 Elsevier Science B.V. All rights reserved.