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Cathepsin L is known as the most unstable lysosomal cysteine proteinase at neutral or alkaline pH. The kinetics of inactivation of human cathepsin L was studied by mixing the enzyme with a substrate and recording the release of product. The inactivation was found to be a first-order process, and the rate of the process decreased with the substrate concentration. The substrate-independent inactivation rate constant kinact was found to be 0.15 s-1 at pH 7.4 and 37 degrees C and increased 85-fold between pH 7.0 and 8.0. At pH 7.4, kinact increased 3200-fold between 5 and 37 degrees C with an energy of activation 174.7 kJ/mol. Inactive cathepsin L did not reactivate at pH 5.5. The rate of inhibition of cathepsin L by stefin B or chicken cystatin at pH 7.4 was much faster than the rate of spontaneous inactivation of the enzyme. The stefin B-cathepsin L complex incubated at pH 7.4 released active enzyme at pH 5.5, suggesting that the cysteine proteinase inhibitors might act as extracellular carriers of the cysteine proteinases.

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Three insect peptides showing high sequence similarity and belonging to the same structural family incorporating a cysteine knot and a short three-stranded antiparallel ␤-sheet were studied. Their inhibitory effect on two serine proteases (bovine ␣-chymotrypsin and human leukocyte elastase) is reported. One of them, PMP-C, is a strong ␣-chymotrypsin inhibitor (K i ‫؍‬ 0.2 nM) and interacts with leukocyte elastase with a K i of 0.12 M. The other two peptides, PMP-D2 and HI, interact only weakly with ␣-chymotrypsin and do not inhibit leukocyte elastase. Synthetic variants of these peptides were prepared by solid-phase synthesis, and their action toward serine proteases was evaluated. This enabled us to locate the P1 residues within the reactive sites (Leu-30 for PMP-C and Arg-29 for PMP-D2 and HI), and, interestingly, variants of PMP-D2 and HI were converted into powerful inhibitors of both ␣-chymotrypsin and leukocyte elastase, the most potent elastase inhibitor obtained in this study having a K i of 3 nM.In the last decade, naturally occurring serine protease inhibitors (1) have been the focus of many studies, mainly for two reasons: first, the target proteins control functions in a variety of fundamental proteolytic processes in humans and mammals (blood clotting, digestion, inflammation, fibrinolysis), in invertebrates such as insects (immune system, digestion, protection against their predators) or worms (protection against their host), and plants (protection against insect attack); second, low molecular weight inhibitors of serine proteases have been attractive tools for studying the general aspects of protein conformation and protein-protein interactions (2). In the present study, we report the inhibitory properties of three homologous peptides (primary and tertiary structures) toward ␣-chymotrypsin, trypsin, and human leukocyte elastase.We have previously isolated two peptides, PMP-C and PMP-D2, from the brain and the fat body of the insect Locusta migratoria (3). These peptides are composed of 36 and 35 residues, respectively, and are cross-linked by three disulfide bonds. The Thr-9 of PMP-C has an uncommon O-glycosidic linkage to a single fucose moiety. There is 40% strict identity between PMP-C and PMP-D2 with conservation of the Cys positions (3). Moreover, they are located on the same peptidic precursor, and, by Northern blot analysis, it has been shown that the gene encoding this precursor is mainly transcribed in the fat body (4). In this paper, we describe the isolation and characterization of HI, a novel locust peptide.Because the isolation from insect extracts is time-consuming and yields only small amounts of peptides, we have prepared, at a reasonable scale (5-10 mg), by solid-phase synthesis, PMP-D2 (5) and PMP-C with and without the fucose moiety. 1Although they are small peptides with a high disulfide content, no sequence similarities could be found when comparing them with small toxins or small protease inhibitors. However, the milligram quantities of PMP-D2 obtained by solid-phase synthesi...

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The kinetics of pH-induced inactivation of human cathepsins B and L was studied by conventional and stopped-flow methods. The inactivation of both enzymes was found to be an irreversible, first-order process. The inactivation rate constants increased exponentially with pH for both enzymes. From log kinac vs pH plots, 3.0 and 1.7 protons were calculated to be desorbed for pH-induced inactivation of cathepsins L and B. Cathepsin B was thus substantially more stable than cathepsin L (approximately 15-fold at pH 7.0 and 37 degrees C). Cathepsin B was efficiently inhibited by cystatin C at pH 7.4, whereas the inhibition by stefin B and high molecular weight kininogen was only moderate. In contrast, cathepsin L was efficiently inhibited by both chicken cystatin and stefin B at this pH kass approximately 3.3 x 10(7) m-1 s-1).

Low molecular mass heparin (5.1 kDa) forms a tight complex with mucus proteinase inhibitor, the physiologic neutrophil elastase inhibitor of the upper respiratory tract. This binding strongly enhances the intrinsic fluorescence of the inhibitor and the rate of neutrophil elastase inhibitor association. One mole of this heparin fragment binds 1 mol of inhibitor with a Kd of 50 nM. From the variation of Kd with ionic strength, it is inferred that (i) 85% of the heparin--inhibitor binding energy i due to electrostatic interactions, (ii) about seven ionic interactions are involved in heparin--inhibitor binding. strength, it is inferred that (i) 85% of the heparin--inhibitor binding energy is due to electrostatic interactions, (ii) about seven ionic interactions are involved in heparin--inhibitor binding. and (iii), about one-third of low quantum yield of Trp30, the single tryptophan residue of the inhibitor, blue-shifts its maximum emission wavelength by 6 nm, decreases the acrylamide quenching rate constant by a factor of 4, and increases the mean intensity weighted lifetime by a factor of 2.5. These important spectroscopic changes evidence a heparin--induced conformational change of the inhibitor which buries Trp30 in a very hydrophobic environment. Heparin accelerates the inhibition of elastase in a concentration-dependent manner. When both enzyme and inhibitor are saturated by the polymer, the second-order association rate constant is 7.7 x 10(7) M-1 s-1, a value that is 27-fold higher than that measured with the free partners. This finding may have important physiologic and therapeutic bearing.