Insight into catalytic mechanism of papain-like cysteine proteinases: the case of D158.

We studied the role of D158 in papain-like cysteine proteinases by using subtilisin Carlsberg, and its chemically modified analog thiolsubtilisin, by applying the proton inventory (PI) method and also by taking into account the pH profiles of the kcat/Km parameter. In the case of thiolsubtilisin, we estimated large inverse solvent isotope effects for kcat/Km, as in papain, whereas for subtilisin we found "dome-shaped" PI, suggesting a completely different mechanism. Finally, the kinetic behavior of thiolsubtilisin presented similarities as well as differences, compared to papain, suggesting a possible role for D158 as part of a catalytic triad in papain-like cysteine proteinases.

[The elastases]. / Les élastases.

Elastases are proteinases capable of solubilizing fibrous elastin. They may belong to the class of serine proteinases, cysteine proteinases and metalloproteinases. Mammalian elastases occur mainly in the pancreas and the phagocytes. Among non-mammalian elastases there is a great variety of bacterial metallo and serine elastases. The elastolytic activity varies from one elastase to another and is usually not correlated with the catalytic efficiency of these proteinases. One may measure this activity using native or labelled elastins. With pure elastases one may use synthetic substrates. There is a large number of natural (proteins) and synthetic elastase inhibitors. Elastases play a pathologic role in pulmonary emphysema, cystic fibrosis, infections, inflammation and atherosclerosis.

The reaction of serpins with proteinases involves important enthalpy changes.

When active serpins are proteolytically inactivated in a substrate-like reaction, they undergo an important structural transition with a resultant increase in their conformational stability. We have used microcalorimetry to show that this conformational alteration is accompanied by an important enthalpy change. For instance, the cleavage of alpha(1)-proteinase inhibitor by Pseudomonas aeruginosa elastase, Staphylococcus aureus V8 proteinase, or papain and that of antithrombin by leukocyte elastase are characterized by large enthalpy changes (DeltaH = -53 to -63 kcal mol(-1)). The former reaction also has a large and negative heat capacity (DeltaC(p)() = -566 cal K(-1) mol(-1)). In contrast, serpins release significantly less heat when they act as proteinase inhibitors. For example, the inhibition of pancreatic elastase, leukocyte elastase, and pancreatic chymotrypsin by alpha(1)-proteinase inhibitor and that of pancreatic trypsin and coagulation factor Xa by antithrombin are accompanied by a DeltaH of -20 to -31 kcal mol(-1). We observe no heat release upon proteolytic cleavage of inactive serpins or following inhibition of serine proteinases by canonical inhibitors or upon acylation of chymotrypsin by N-trans-cinnamoylimidazole. We suggest that part of the large enthalpy change that occurs during the structural transition of serpins is used to stabilize the proteinase in its inactive state.

Insight into the catalysis of hydrolysis of four newly synthesized substrates by papain: a proton inventory study.

We synthesized the following four new peptide substrates, Suc-Phe-Leu-pNA, Suc-Phe-Leu-NMec, Suc-Phe-Leu-ONPh, and Pht-Phe-Leu-pNA, and we applied the proton inventory method to their hydrolysis by papain. Useful relationships between the rate constants of the catalytic reaction have been established and contributed to the elucidation of the hydrolytic mechanism of papain. For all amide substrates, the parameter K(S) and the rate constants k(1), k(-)(1), and k(2) were estimated. Moreover, it was found that k(cat)/K(m) = k(1) for all four substrates, while two exchangeable hydrogenic sites, one in the ground state and another in the transition state, generate an inverse isotope effect during the reaction governed by this parameter. The proton inventories of both k(2) and k(3) are essentially linear, whatever the acyl moiety and/or the leaving group of the substrate. The proton inventories of K(S) are also essentially linear for all amide substrates, while the observed large isotope effect of about 3 to 9 originates from a single hydrogenic site in the product state. This latter, in agreement to both the small transition state fractionation factors found for k(cat)/K(m) (or k(1)) and the unit ground-state fractionation factors found for k(2), argues for the formation of a tetrahedral adduct during the reaction governed by the k(1) parameter. Furthermore, papain acts as a one-proton catalyst during acylation or deacylation, both of which proceed through similar concerted reaction pathways, where a nucleophilic attack is accompanied by the movement of one proton.

Effect of DNase on the activity of neutrophil elastase, cathepsin G and proteinase 3 in the presence of DNA.

It has been shown previously that DNA binds and inhibits neutrophil elastase (NE). Here we demonstrate that DNA has a better affinity for neutrophil cathepsin G (cat G) than for NE and is a better inhibitor of cat G than of NE. DNase-generated <0.5 kb DNA fragments inhibit NE and cat G as potently as full length DNA. This rationalises our observation that administration of DNase to cystic fibrosis patients does not enhance the NE and cat G activity of their lung secretions. Neutrophil proteinase 3 is not inhibited by DNA and might thus be the most harmful proteinase in inflammatory lung diseases.

DNA strongly impairs the inhibition of cathepsin G by alpha(1)-antichymotrypsin and alpha(1)-proteinase inhibitor.

This paper explores the possibility that neutrophil-derived DNA interferes with the inhibition of neutrophil cathepsin G (cat G) and proteinase 3 by the lung antiproteinases alpha(1)-proteinase inhibitor (alpha(1)PI), alpha(1)-antichymotrypsin (ACT), and mucus proteinase inhibitor (MPI). A 30-base pair DNA fragment ((30bp)DNA), used as a model of DNA, tightly binds cat G (K(d), 8.5 nM) but does not react with proteinase 3, alpha(1)PI, ACT, and MPI at physiological ionic strength. The polynucleotide is a partial noncompetitive inhibitor of cat G whose K(i) is close to K(d). ACT and alpha(1)PI are slow binding inhibitors of the cat G-(30bp)DNA complex whose second-order rate constants of inhibition are 2300 M(-1) s(-1) and 21 M(-1) s(-1), respectively, which represents a 195-fold and a 3190-fold rate deceleration. DNA thus renders cat G virtually resistant to inhibition by these irreversible serpins. On the other hand, (30bp)DNA has little or no effect on the reversible inhibition of cat G by MPI or chymostatin or on the irreversible inhibition of cat G by carbobenzoxy-Gly-Leu-Phe-chloromethylketone. The polynucleotide neither inhibits proteinase 3 nor affects its rate of inhibition by alpha(1)PI. These findings suggest that cat G may cause lung tissue destruction despite the presence of antiproteinases.

Evidence that translocation of the proteinase precedes its acylation in the serpin inhibition pathway.

The inhibition of proteinases by serpins involves cleavage of the serpin, acylation, and translocation of the proteinase. To see whether acylation precedes or follows translocation, we have investigated the pH dependence of the interaction of fluorescein isothiocyanate-elastase with rhodamine alpha(1)-proteinase inhibitor (alpha(1)PI) using two independent methods: (i) kinetics of fluorescence energy transfer which yields k(2,f), the rate constant for the fluorescently detected decay of the Michaelis-type complex (Mellet, P., Boudier, C., Mély, Y., and Bieth, J. G. (1998) J. Biol. Chem. 273, 9119-9123); (ii) kinetics of elastase-catalyzed hydrolysis of a substrate in the presence of alpha(1)PI, which yields k(2,e), the rate constant for the conversion of the Michaelis-type complex into irreversibly inhibited elastase. Both rate constants were found to be pH-independent and close to each other, indicating that acylation, a pH-dependent phenomenon, does not govern the decay of the Michaelis-type complex and, therefore, follows translocation. On the other hand, anhydro-elastase reacts with alpha(1)PI to form a Michaelis-type complex that translocates into a second complex with a rate constant close to that measured with active elastase, confirming that acylation is not a prerequisite for translocation. Moreover, the anhydro-elastase-alpha(1)PI complex was found to be thermodynamically reversible, suggesting that translocation of active elastase might also be reversible. We propose that serpins form a Michaelis-type complex EI(M), which reversibly translocates into EI(tr) whose acylation yields the irreversible complex EI(ac). [see text]

Inhibition of neutrophil cathepsin G by oxidized mucus proteinase inhibitor. Effect of heparin.

Oxidation of mucus proteinase inhibitor (MPI) transforms Met73, the P'1 residue of its active center into methionine sulfoxide and lowers its affinity for neutrophil elastase [Boudier, C., and Bieth, J. G. (1994) Biochem. J. 303, 61-68]. Here, we show that the oxidized inhibitor has also a decreased affinity for neutrophil cathepsin G and pancreatic chymotrypsin. The Ki of the oxidized MPI-cathepsin G complex (1.2 microM) is probably too high to be compatible with significant inhibition of cathepsin G in inflammatory lung secretions. Stopped-flow kinetics shows that, within the inhibitor concentration range used, the mechanism of inhibition of cathepsin G and chymotrypsin by oxidized MPI is consistent with a one-step reaction, [equation in text] whereas the inhibition of elastase takes place in two steps, [equation in text]. Heparin, which accelerates the inhibition of the three proteinases by native MPI, also favors their interaction with oxidized MPI. Flow calorimetry shows that heparin binds oxidized MPI with Kd, Delta H degrees, and Delta S degrees values close to those reported for native MPI. In the presence of heparin, oxidized MPI inhibits cathepsin G via a two-step reaction characterized by Ki = 0.22 microM, k2 = 0.1 s-1, k-2 = 0.023 s-1, and Ki = 42 nM. Under these conditions, in vivo inhibition of cathepsin G is again possible. Heparin also improves the inhibition of chymotrypsin and elastase by oxidized MPI by increasing their kass or k2/Ki and decreasing their Ki. Our data suggest that oxidation of MPI during chronic bronchitis may lead to cathepsin G-mediated lung tissue degradation and that heparin may be a useful adjuvant of MPI-based therapy of acute lung inflammation in cystic fibrosis.

Acidic pH as a physiological regulator of human cathepsin L activity.

Human cysteine protease cathepsin L was inactivated at acid pH by a first-order process. The inactivation rate decreased with increasing concentrations of a small synthetic substrate, suggesting that substrates stabilize the active conformation. The substrate-independent inactivation rate constant increased with organic solvent content of the buffer, consistent with internal hydrophobic interactions, disrupted by the organic solvent, also stabilizing the enzyme. Circular dichroism showed that the inactivation is accompanied by large structural changes, a decrease in alpha-helix content being especially pronounced. The high activation energy of the reaction at pH 3.0 (200 kJ.mol-1) supported such a major conformational change occurring. The acid inactivation of cathepsin L was irreversible, consistent with the propeptide being needed for proper folding of the enzyme. Aspartic protease cathepsin D was shown to cleave denatured, but not active cathepsin L, suggesting a potential mechanism for in-vivo regulation and turnover of cathepsin L inside lysosomes.

Effect of polynucleotides on the inhibition of neutrophil elastase by mucus proteinase inhibitor and alpha 1-proteinase inhibitor.

DNA released from neutrophils at sites of inflammation may modulate tissue proteolysis. We used tRNA and synthetic polynucleotides as models of DNA to study the influence of polynucleotides on the inhibition of neutrophil elastase by its endogenous inhibitors alpha1-proteinase inhibitor (alpha1-PI) and mucus proteinase inhibitor (MPI). Affinity chromatography showed that polynucleotides form electrostatic complexes with elastase and MPI but not with alpha1-PI, the highest affinity being for MPI. The tight-binding partial inhibition of elastase by polynucleotides was used to calculate the Kd of the elastase-polynucleotide complexes which ranged from 4 microM to 21 nM. One mole of tRNA was able to bind 9 mol of elastase. Polydeoxycytosine and tRNA significantly impaired the reversible inhibition of elastase by MPI: they moderately increased the rate of enzyme-inhibitor association, strongly enhanced the rate of complex dissociation, and lowered the enzyme-inhibitor affinity by factors of 34 and 134, respectively. The two polynucleotides also decreased the rate of the irreversible inhibition of elastase by alpha1-PI by factors of 30 and 3, respectively. Polynucleotides also changed the mechanism of inhibition of elastase by the two inhibitors from a one-step inhibition reaction to a two-step binding mechanism. Our data may help explain why proteolysis may occur at sites of inflammation despite the presence of active proteinase inhibitors.

Kinetic mechanism of the inhibition of cathepsin G by alpha 1-antichymotrypsin and alpha 1-proteinase inhibitor.

Uncontrolled proteolysis due to cathepsin G (cat G) may cause severe pathological disorders. Cat G is inhibited by alpha 1-antichymotrypsin (ACT) and alpha 1-proteinase inhibitor (alpha 1PI), two members of the serpin superfamily of proteins. To see whether these two inhibitors play a physiological proteolysis-preventing function, we have made a detailed kinetic investigation of their reaction with cat G. The kinetics of inhibition of cat G in the presence of inhibitor and substrate evidenced a two-step inhibition mechanism: E + I EI EI. The cat G/ACT interaction is described by Ki = 6.2 x 10(-)8 M and k2 = 2.8 x 10(-)2 s-1, while the cat G/alpha 1PI association is governed by Ki = 8.1 x 10(-)7 M and k2 = 5.5 x 10(-)2 s-1. The reliability of these kinetic constants was checked using a number of experiments which all gave consistent results: (i) both EI complexes were found to be enzymatically inactive, (ii) the Ki values were determined directly using initial velocity experiments of cat G-catalyzed hydrolysis of substrate in the presence of inhibitor, (iii) the second-order rate constants k2/Ki were measured using second-order inhibition experiments in the absence of substrate, and (iv) the ratio of the two second-order rate constants was determined by measuring the partition of cat G between the two fluorescently labeled serpins. Since the plasma concentrations of ACT and alpha 1PI are much higher than their Ki values, cat G released from neutrophils will be fully taken up as rapidly forming EI complexes, that is, 70% with ACT and 30% with alpha 1PI. Both ACT and alpha 1PI are thus physiological cat G inhibitors whose inhibitory potential does not depend on the formation of the stable inhibitory species EI characteristic of serpins. Such an in vivo inhibition mechanism might take place with other serpin/proteinase systems.

Stopped flow fluorescence energy transfer measurement of the rate constants describing the reversible formation and the irreversible rearrangement of the elastase-alpha1-proteinase inhibitor complex.

Serpins are thought to inhibit proteinases by first forming a Michaelis-type complex that later converts into a stable inhibitory species. However, there is only circumstantial evidence for such a two-step reaction pathway. Here we directly observe the sequential appearance of two complexes by measuring the time-dependent change in fluorescence resonance energy transfer between fluorescein-elastase and rhodamine-alpha1-protease inhibitor. A moderately tight initial Michaelis-type complex EI1 (Ki = 0.38-0.52 microM) forms and dissociates rapidly (k1 = 1.5 x 10(6) M-1 s-1, k-1 = 0.58 s-1). EI1 then slowly converts into EI2 (k2 = 0.13 s-1), the fluorescence intensity of which is stable for at least 50 s. The two species differ by their donor-acceptor energy transfer efficiency (0. 41 and 0.26, respectively). EI2 might be the final product of the elastase + inhibitor association because its transfer efficiency is the same as that of a complex incubated for 30 min. The time-dependent change in fluorescence resonance energy transfer between fluorescein-elastase and rhodamine-eglin c, a canonical inhibitor, again allows the fast formation of a complex to be observed. However, this complex does not undergo any fluorescently detectable transformation.

Heparin accelerates the inhibition of cathepsin G by mucus proteinase inhibitor: potent effect of O-butyrylated heparin.

Heparin tightly binds cathepsin G and so protects the enzyme from inhibition by alpha1-antichymotrypsin, alpha1-proteinase inhibitor and eglin c, three proteins which do not bind heparin [Ermolieff J., Boudier C., Laine A., Meyer B. and Bieth J.G. (1994) J. Biol. Chem. 269, 29502-29508]. Here we show that heparin no longer protects cathepsin G from inhibition when the enzyme is reacted with mucus proteinase inhibitor (MPI), a heparin-binding protein. Heparin fragments of Mr=4500 and 8100 and O-butyrylated heparin of Mr=8000 form tight complexes with cathepsin G (Kd=0.5-2.2 nM) and MPI (Kd=0. 4-0.8 muM) and accelerate the MPI-promoted inhibition of cathepsin G by a factor of 17-26. They also accelerate the inhibition of neutrophil elastase and pancreatic chymotrypsin. The rate acceleration is due to the binding of heparin to MPI. Butyrylation of heparin slightly decreases its affinity for cathepsin G and MPI but sharply decreases the ionic interactions between the positively charged proteins and the negatively charged polyanion. The butyrylated heparin derivative is the best rate accelerator: it increases the rate constant for the MPI-induced inhibition of cathepsin G and elastase by factors of 26 and 23, respectively. This, together with the fact that it has a good bioavailability and a very low anticoagulant activity, suggests that it might be an adjuvant of MPI-based therapy of cystic fibrosis.

Inhibition of neutrophil serine proteinases by suramin.

Suramin, a hexasulfonated naphtylurea recently used as an anti-tumor drug, is a potent inhibitor of human neutrophil elastase, cathepsin G, and proteinase 3. The complexes it forms with these enzymes are partially active on synthetic substrates, but full inhibition takes place when elastase activity is measured with fibrous elastin or when cathepsin G activity is measured using platelet aggregation. One molecule of elastase binds four molecules of suramin with a Ki of 2 x 10(-7) M as determined by enzyme inhibition or intrinsic fluorescence enhancement of suramin. The binding curves show no sign of cooperativity or anticooperativity. The Ki for the complexes with cathepsin G and proteinase 3 are 8 x 10(-8) and 5 x 10(-7) M, respectively. Ionic strength increases the Ki of the elastase-suramin complex in a way that suggests that four of the six sulfonate groups of suramin form ionic interactions with basic residues of the enzyme and that at saturation almost all arginines of elastase form salt bridges with suramin. The neutrophil proteinase-inhibitory activity of suramin might be used to prevent tissue destruction and thrombus formation in diseases where massive infiltration and activation of neutrophils take place.

Mapping the suramin-binding sites of human neutrophil elastase: investigation by fluorescence resonance energy transfer and molecular modeling.

Neutrophil elastase (NE), a mediator of inflammation, binds with high affinity numerous anionic molecules including suramin, a polysulfated naphthylurea, which inhibits it with a Ki of 0.2 microM and a 4:1 suramin:NE stoichiometry and thus constitutes a potential therapeutic agent. In an attempt to locate the suramin molecules on NE, we investigated the NE-suramin interaction using steady-state and time-resolved fluorescence spectroscopy. The time-resolved intensity decay of NE, a protein with three Trp residues, in positions 27, 141, and 237 (chymotrypsin numbering system) was best described by a three-exponential function with lifetimes ranging from 0.22 to 2.28 ns. Comparison of the accessibility of the three lifetime classes to the fluorescence quenchers acrylamide and iodide with the computed solvent accessibility of the three Trp residues in the crystal structure of NE indicates that the main, if not the sole, contribution to the 2.28 ns lifetime class is brought about by the fully buried Trp 141 residue. The addition of suramin to NE induces a sharp decrease in NE fluorescence and a corresponding increase in suramin fluorescence due to an efficient fluorescence resonance energy transfer (FRET) between the Trp residues of NE, acting as donors, and the naphthalene rings of suramin, behaving as acceptors. From the fate of the longest lifetime class in the presence of variable suramin concentrations, we deduce that two suramins are bound at less than 17 A from Trp 141, whereas the two others are located at least 29 A from Trp 141. Moreover, neither the binding of suramin to NE nor the FRET process was modified when NE was complexed with a peptide chloromethylketone inhibitor, suggesting that suramin does not directly interfere with the substrate binding site of NE. These data were used as constraints to model the NE-suramin complex.

Heat-induced conversion of ovalbumin into a proteinase inhibitor.

Ovalbumin is a member of the serine proteinase inhibitor (serpin) family but is unable to inhibit proteinases. Here we show that heating transforms it into inhibitory ovalbumin (I-ovalbumin), a potent reversible competitive inhibitor of human neutrophil elastase (Ki = 5 nM) and cathepsin G (Ki = 60 nM) and bovine chymotrypsin (Ki = 30 nM). I-ovalbumin also inhibits bovine trypsin, porcine elastase and alpha-lytic proteinase with Ki values in the micromolar range. Thus, I-ovalbumin differs from active serpins by its inability to form irreversible complexes with proteinases. I-ovalbumin is unusually thermostable: it does not undergo any structural transition between 45 degrees C and 120 degrees C as tested by differential scanning calorimetry, and it retains full inhibitory capacity after heating at 120 degrees C. It has 8% less alpha-helices and 9% more beta-sheet structures than native ovalbumin, as shown by circular dichroism. Our results show that the primary sequence of ovalbumin contains the information required for enabling the first step of the serpin-proteinase interaction to occur, i.e. the formation of the Michaelis-like reversible complex, but does not contain the information needed for stabilizing this initial complex.

Inhibition of neutrophil elastase by the alpha1-proteinase inhibitor-immunoglobulin A complex.

Neutrophil elastase is thought to be involved in cartilage destruction occurring in rheumatoid arthritis despite the local presence of alpha1-proteinase inhibitor. Part of synovial fluid alpha1-proteinase inhibitor forms a mixed disulfide with immunoglobulin A, which has been postulated to lack inhibitory activity. We show here that the immunoglobulin-inhibitor complex tightly inhibits neutrophil elastase and cathepsin G, bovine pancreatic trypsin and chymotrypsin, and porcine pancreatic elastase. Although the rate constant of inhibition of neutrophil elastase by immunoglobulin A-bound alpha1-proteinase inhibitor (k(ass) = 9.2 X 10(5) M(-1) x s(-1)) is about 10-fold lower than that measured with the free inhibitor, it is high enough to enable efficient inhibition of elastase in vivo.

Inhibition of human pancreatic proteinases by mucus proteinase inhibitor, eglin c and aprotinin.

The kinetic investigation of the inhibition of human pancreatic trypsin 1, trypsin 2 and chymotrypsin A by mucus proteinase inhibitor, eglin c and aprotinin reveals that (i) the first protein is a potent inhibitor of chymotrypsin A (kass. = 1.4 x 10(6) M-1.s-1, Ki = 71 pM) but forms loose complexes with trypsin 1 (Ki = 0.5 microM) and trypsin 2 (Ki = 18 nM), (ii) eglin c does not inhibit the two trypsins but forms a tight complex with chymotrypsin A (kass. = 3.3 x 10(6) M-1.s-1, Ki < 0.1 nM) and (iii) aprotinin is a potent inhibitor of trypsin 1 (kass. = 1 x 10(6) M-1.s-1, Ki < 0.2 nM) and trypsin 2 (kass. = 2.4 x 10(5) M-1.s-1, Ki < 1 nM) but forms a loose complex with chymotrypsin A (Ki = 0.17 microM). These data, together with those published previously on human pancreatic elastase, suggest that a cocktail of aprotinin + eglin c might be a better intensive-care drug for acute pancreatitis than aprotinin alone, because it will efficiently inhibit all four human pancreatic proteinases. On the other hand, human gastric juice inactivates mucus proteinase inhibitor by pepsin-mediated cleavage. This indicates that the fraction of mucus proteinase inhibitor that reaches the stomach following aerosol delivery to cystic fibrosis patients does not reach the duodenum in an active form and, therefore, does not aggravate the pancreatic insufficiency of these patients.

Serine protease inhibition by insect peptides containing a cysteine knot and a triple-stranded beta-sheet.

Three insect peptides showing high sequence similarity and belonging to the same structural family incorporating a cysteine knot and a short three-stranded antiparalled beta-sheet were studied. Their inhibitory effect on two serine proteases (bovine alpha-chymotrypsin and human leukocyte elastase) is reported. One of them, PMP-C, is a strong alpha-chymotrypsin inhibitor (Ki = 0.2 nM) and interacts with leukocyte elastase with a Ki of 0.12 microM. The other two peptides, PMP-D2 and HI, interact only weakly with alpha-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 alpha-chymotrypsin and leukocyte elastase, the most potent elastase inhibitor obtained in this study having a Ki of 3 nM.

High-affinity binding of two molecules of cysteine proteinases to low-molecular-weight kininogen.

Human low-molecular-weight kininogen (LK) was shown by fluorescence titration to bind two molecules of cathepsins L and S and papain with high affinity. By contrast, binding of a second molecule of cathepsin H was much weaker. The 2:1 binding stoichiometry was confirmed by titration monitored by loss of enzyme activity and by sedimentation velocity experiments. The kinetics of binding of cathepsins L and S and papain showed the two proteinase binding sites to have association rate constants kass,1 = 10.7-24.5 x 10(6) M-1 s-1 and kass,2 = 0.83-1.4 x 10(6) M-1 s-1. Comparison of these kinetic constants with previous data for intact LK and its separated domains indicate that the faster-binding site is also the tighter-binding site and is present on domain 3, whereas the slower-binding, lower-affinity site is on domain 2. These results also indicate that there is no appreciable steric hindrance for the binding of proteinases between the two binding sites or from the kininogen light chain.