Streptomyces griseus protease B: secretion correlates with the length of the propeptide

Streptomyces griseus protease B, a member of the chymotrypsin superfamily, is encoded by a gene that express a pre-pro-mature protein. During secretion the precursor protein is processed into a mature, fully folded protease. In this study, we constructed a family of genes which encode deletions at the amino-terminal end of the propeptide. The secretion of active protease B was seen to decrease in an exponential manner according to the length of the deletion. The results underscore the intimate relationship between folding and secretion in bacterial protease expression. They further suggest that the propeptide segment of the zymogen stabilizes the folding of the mature through many small binding interactions over the entire surface of the peptide rather than through a few specific contacts.     

Human prochymase activation. A novel role for heparin in zymogen processing

Human prochymase is packaged with heparin in mast cell granules and appears to be activated by dipeptidylpeptidase I. We show that a high affinity interaction between heparin and prochymase allows the 2-residue propeptide to be cleaved by dipeptidylpeptidase I. A conserved Glu in the propeptide is necessary for this heparin effect. Following propeptide cleavage, capture of the newly generated NH2 terminus by an "activation groove" on the enzyme activates the enzyme and concurrently prevents a progressive degradation of the NH2 terminus by dipeptidylpeptidase I. Surrogate peptide studies show that the activation groove is unoccupied in prochymase and is specific for the chymase NH2 terminus. These observations indicate that heparin is an important cofactor in the prochymase activation process and explain how dipeptidylpeptidase I, a nonspecific processing enzyme, can effect a specific cleavage of the zymogen propeptide.     

Granzyme B mimics apical caspases. Description of a unified pathway for trans-activation of executioner caspase-3 and -7

Granzyme B (GrB) is predicted to trigger apoptosis by activating preferred caspases, but the zymogens that are directly processed by the granzyme and the requirements for these interactions remain unclarified. We examined this dilemma by comparing the kinetics and pattern of GrB-mediated activation of the executioner caspase-7 in vitro and in vivo. GrB rapidly activates procaspase-7 in vitro by cleaving between the large and small subunits leaving the propeptide intact. During GrB-mediated apoptosis, the caspase-7 propeptide is removed and cleavage occurs between the subunits. Strikingly, caspase-7 is unprocessed in caspase-3-deficient MCF-7 cells exposed to GrB but is rapidly activated when the cells are solubilized. Transfection with caspase-3 restores the removal of the caspase-7 propeptide and the capacity of GrB to subsequently activate the caspase. The data suggest that GrB activates caspase-3, which then removes the propeptide of caspase-7 allowing activation by GrB. Thus GrB initiates the death pathway by processing the accessible caspase-3, and the caspase-7 propeptide regulates trans-activation of the zymogen by granzyme. As a consequence, two proteases, caspase-3 and GrB, are required to activate procaspase-7.     

Variants of tissue-type plasminogen activator which display substantially enhanced stimulation by fibrin

Unlike most proteases, tissue-type plasminogen activator (t-PA) is secreted from cells as an active, single chain "proenzyme" whose catalytic efficiency is comparable with that of the corresponding mature, two-chain enzyme. We have previously suggested that the absence of the "zymogen triad" (Asp194-His40-Ser32; chymotrypsin numbering) contributes to this unusually high enzymatic activity of single chain t-PA. Consistent with this prediction, the single chain form of a variant of t-PA containing the zymogen triad displayed dramatically reduced activity toward synthetic substrates. Activation cleavage of this variant, however, resulted in a mature, two-chain enzyme with full catalytic activity. To further examine the functional significance of the zymogen triad, we used site-specific mutagenesis to construct a variant of t-PA, t-PA/R275E,A292S,F305H, that contained this triad but could not be converted into its two-chain form by plasmin. Characterization of this variant demonstrated that the presence of the zymogen triad specifically suppressed plasminogen activation by single chain t-PA in the absence of fibrin. In addition, these studies indicated that, like wild type t-PA, zymogen activation of this variant could be accomplished by binding to the co-factor fibrin. The combination of full activity in the presence of fibrin and reduced activity in its absence resulted in novel variants of t-PA that displayed dramatically enhanced stimulation by fibrin. While the presence of fibrin increased the catalytic efficiency of t-PA toward plasminogen by a factor of approximately 520, this stimulation factor increased to 130,000 for t-PA/R275E,A292S,F305H. Plasmin-resistant, zymogen-like variants of t-PA, therefore, may represent thrombolytic enzymes with enhanced "clot selectivity."     

Properties of the His57-Asp102 dyad of rat trypsin D189S in the zymogen, activated enzyme, and alpha1-proteinase inhibitor complexed forms

Structural and biochemical studies suggest that serpins induce structural rearrangements in their target serine-proteinases. Previous NMR studies of the complex between a serpin, alpha1-proteinase inhibitor, and a mutant of recombinant rat trypsin (the Asp189 to Ser mutant, D189S, which is much more stable than wild-type rat trypsin against autoproteolysis) provided information about the state of catalytic residues in this complex: the hydrogen bond between Asp102 and His57 remains intact in the complex, and spectral properties of His57 are more like those of the zymogen than of the activated enzyme (G. Kaslik, et al., 1997, Biochemistry 36, 5455-5464). Here we report the protonation and exchange behavior of His57 of recombinant rat trypsin D189S in three states: the zymogen, the active enzyme, and the complex with human alpha1-proteinase inhibitor and compare these with analogous behavior of His57 of bovine chymotrypsinogen and alpha-chymotrypsin. In these studies the pKa of His57 has been determined from the pH dependence of the 1H NMR signal from the Hdelta1 proton of histidine in the Asp102-His57 dyad, and a measure of the accessibility of this part of the active site has been obtained from the rate of appearance of this signal following its selective saturation. The activation of rat trypsinogen D189S (zymogen, pKa = 7.8 +/- 0.1; Hill coefficient = 0. 86 +/- 0.05) decreased the pKa of His57 by 1.1 unit and made the protonation process cooperative (active enzyme, pKa = 6.7 +/- 0.1; Hill coefficient = 1.37 +/- 0.08). The binding of alpha1-proteinase inhibitor to trypsin D189S led to an increase in the pKa value of His57 to a value higher than that of the zymogen and led to negative cooperativity in the protonation process (complex, pKa = 8.1 +/- 0. 1; Hill coefficient = 0.70 +/- 0.08), as was observed for the zymogen. In spite of these differences in the pKa of His57 in the zymogen, active enzyme, and alpha1-proteinase inhibitor complex, the solvent exchange lifetime of the His57 Hdelta1 proton was the same, within experimental error, in all three states (lifetime = 2 to 12.5 ms). The linewidth of the 1H NMR signal from the Hdelta1 proton of His57 was relatively sharp, at temperatures between 5 and 20 degrees C at both low pH (5.2) and high pH (10.0), in spectra of bovine alpha-chymotrypsin, recombinant rat trypsin D189S, and the complex between rat trypsin D189S and human alpha1-proteinase inhibitor; however, in spectra of the complex between alpha-chymotrypsin and human alpha1-proteinase inhibitor, the peak was broader and could be well-resolved only at the lower temperature (5 degrees C).     

Characterization of the precursor of prostate-specific antigen. Activation by trypsin and by human glandular kallikrein

The precursor or zymogen form of prostate-specific antigen (pro-PSA) is composed of 244 amino acid residues including an amino-terminal propiece of 7 amino acids. Recombinant pro-PSA was expressed in Escherichia coli, isolated from inclusion bodies, refolded, and purified. The zymogen was readily activated by trypsin at a weight ratio of 50:1 to generate PSA, a serine protease that cleaves the chromogenic chymotrypsin substrate 3-carbomethoxypropionyl-L-arginyl-L-prolyl-L-tyrosine-p-nitroanili ne- HCl (S-2586). In this activation, the amino-terminal propiece Ala-Pro-Leu-Ile-Leu-Ser-Arg was released by cleavage at the Arg-Ile peptide bond. The recombinant pro-PSA was also activated by recombinant human glandular kallikrein, another prostate-specific serine protease, as well as by a partially purified protease(s) from seminal plasma. The recombinant PSA was inhibited by alpha1-antichymotrypsin, forming an equimolar complex with a molecular mass of approximately 100 kDa. The recombinant PSA failed to activate single chain urokinase-type plasminogen activator, in contrast to the recombinant hK2, which readily activated single chain urokinase-type plasminogen activator. These results indicate that pro-PSA is converted to an active serine protease by minor proteolysis analogous to the activation of many of the proteases present in blood, pancreas, and other tissues. Furthermore, PSA is probably generated by a cascade system involving a series of precursor proteins. These proteins may interact in a stepwise manner similar to the generation of plasmin during fibrinolysis or thrombin during blood coagulation.     

Cutting at the right place--the importance of selective limited proteolysis in the activation of proproteinase E

Proteinase E is a proteolytic enzyme which belongs to a distinct subfamily of chymotrypsin-like serine endopeptidases. Its proform from the bovine pancreatic system has been structurally analyzed by X-ray crystallography for the intact native form, with a 11-residue N-terminal activation peptide, in a ternary complex with chymotrypsinogen C and procarboxypeptidase A [Gomis-Rüth, F. X., Gómez, M., Bode, W., Huber, R. & Avilés, F. X. (1995) The three-dimensional structure of the native ternary complex of bovine pancreatic procarboxypeptidase A with proproteinase E and chymotrypsinogen C, EMBO J. 14, 4387-4394]. Also for a N-terminally truncated form, lacking the first 13 residues and called subunit III, a crystal structure is available [Pignol, D., Gaboriaud, C., Michon, T., Kerfelec, B., Chapus, C. & Fontecilla-Camps, J. C. (1994) Crystal structure of bovine procarboxypeptidase A-S6 subunit III, a highly structured truncated zymogen E, EMBO J. 8, 1763-1771]. Both structures are well defined by electron density, except for the first 7 residues of subunit III. However, both structures present large deviations of up to 2 nm in several regions, indicating that they correspond to two quite distinct states of low free energy, influenced by very few contacts made via the N-terminal segment. As no structure of an active proteinase E is known so far, pancreatic porcine elastase has been chosen as a model for this enzyme and an activation mechanism for this distinct serine endopeptidase subfamily is proposed.     

Pneumonia after heart transplantation: a multi-institutional study. Spanish Transplantation Infection Study Group

Matrix metalloproteinases (MMPs) are involved in connective tissue turnover under physiological and pathological conditions. MMP activity is regulated by the requirement for zymogen activation. This report describes a proMMP-3 activator produced by articular cartilage. The activator initiates a step-wise processing of proMMP-3 to generate an array of active species. Sequencing of activation intermediates demonstrated cleavage on the NH2-terminal side of certain basic residues in the MMP-3 propeptide. Metal ion chelators inhibited activator-dependent proteolysis, and activity was restored by low levels of ZnCl2. These catalytic properties suggest similarity to members of the insulinase superfamily of metalloendopeptidases with in vitro specificity for single arginine or paired basic processing sites in a variety of prohormones. Dibasic sites also exist in the propeptides of several MMPs including proMMP-3. This is the first report that cartilage produces a potent MMP proenzyme activator, opening the possibility of a novel intrinsic activation pathway for catabolic processes in this avascular tissue.     

APMA (4-aminophenylmercuric acetate) activation of stromelysin-1 involves protein interactions in addition to those with cysteine-75 in the propeptide

Matrix metalloproteinases (MMPs) can be activated in vitro by multiple mechanisms such as treatment with proteases, organomercurials, oxidants, and detergents. The proposed cysteine switch model for activation suggests that these multiple methods for activation cause the dissociation of the single cysteine residue in the propeptide from the active site zinc. In particular, it has been suggested that organomercurials such as 4-aminophenylmercuric acetate (APMA) work by directly reacting with the sulfhydryl group of this cysteine residue, resulting in its displacement from the active site. However, recent data by Chen et al. [(1993) Biochemistry 32, 10289-10295] demonstrated that modification of this cysteine residue in the propeptide of stromelysin-1 by sulfhydryl reagents did not result in an active enzyme as predicted. To investigate the roles that this cysteine residue and the propeptide salt bridge (R74 to D79) might play in the APMA-induced activation of stromelysin-1, we have changed these residues by site-directed mutagenesis. Wild-type stromelysin-1 and the mutants were all expressed at detectable levels using a recombinant vaccinia virus system and determined to be catalytically competent by zymography. The wild-type stromelysin-1 and the cysteine mutants (C75S and C75H) underwent APMA-induced activation as determined by the characteristic reduction in molecular weight associated with activation and by their ability to cleave casein only when activated. On the other hand, mutants R74K, D79A, and C75H/D79A did not undergo APMA-induced activation. These results demonstrate that APMA-induced activation of stromelysin-1 involves protein interactions in addition to those with cysteine-75 in the propeptide and also suggest that the R74 to D79 salt bridge may play a role.     

An induced proximity model for caspase-8 activation

The assembly of the CD-95 (Fas/Apo-1) receptor death-inducing signaling complex occurs in a hierarchical manner; the death domain of CD-95 binds to the corresponding domain in the adapter molecule Fas-associated death domain (FADD) Mort-1, which in turn recruits the zymogen form of the death protease caspase-8 (FLICE/Mach-1) by a homophilic interaction involving the death effector domains. Immediately after recruitment, the single polypeptide FLICE zymogen is proteolytically processed to the active dimeric species composed of large and small catalytic subunits. Since all caspases cleave their substrates after Asp residues and are themselves processed from the single-chain zymogen to the two-chain active enzyme by cleavage at internal Asp residues, it follows that an upstream caspase can process a downstream zymogen. However, since FLICE represents the most apical caspase in the Fas pathway, its mode of activation has been enigmatic. We hypothesized that the FLICE zymogen possesses intrinsic enzymatic activity such that when approximated, it autoprocesses to the active protease. Support for this was provided by (i) the synthesis of chimeric Fpk3FLICE molecules that can be oligomerized in vivo by the synthetic cell-permeable dimerizer FK1012H2. Cells transfected with Fpk3FLICE underwent apoptosis after exposure to FK1012H2; (ii) the creation of a nonprocessable zymogen form of FLICE that retained low but detectable protease activity.     

Spontaneous propeptide processing of mini-stromelysin-1 mutants blocked by APMA ((4-Aminophenyl)mercuric acetate)

Human stromelysin-1 (SL-1) is a member of the stromelysin subfamily of matrix metalloproteinases (MMPs). The MMPs play a major role in the degradation of the extracellular matrix (ECM) during normal and pathological conditions. SL-1 like the other MMPs can be activated in vitro by the stepwise removal of the propeptide that contains a single unpaired cysteine which coordinates the active site zinc. Other residues in the propeptide also play a role in maintaining the latency of the enzymes. Deletion mutants and single-site amino acid replacements within the propeptide of a carboxyl-terminally truncated stromelysin-1 (mini-SL-1) were constructed and expressed in Escherichia coli to further examine what amino acids within the propeptide of SL-1 are important for maintaining latency. While the natural enzyme displayed some limited tendency to spontaneously (autolytically) convert to lower Mr in a stepwise manner and finally to the fully processed form, all of the truncation mutants of more than 19 amino acids generated in E. coli showed greatly accelerated self-cleavage indicative of diminished stability and/or resistance to proteolysis of the residual propeptide. Mutant Delta63 as well as other mutants in which most of the propeptide had been deleted no longer responded to exposure to the organomercurial APMA by accelerated autolytic processing. Rather, APMA inhibited the autolytic processing in these mutants, further confirming the complexity of the action of this organomercurial in the activation of pro-MMPs.     

Processing of procathepsin from Musca domestica eggs

The major source of amino acids for insect embryos are yolk proteins which accumulate in developing oocytes and are hydrolyzed during embryogenesis. Studies on Musca domestica embryogenesis indicated that a cathepsin B-like proteinase is responsible for yolk protein degradation (Ribolla et al., 1993). In this study, we report the purification of mature cathepsin and show that it is made up of a single 41 kDa polypeptide chain. The Musca domestica cathepsin NH2-terminal 11-residue sequence was determined (Ala-Pro-Lys-Tyr-Val-Asp-Tyr-Gly-Glu-Asn-Glu) and reveals homology with other cathepsins of the papain family. Experiments using serum anti-cathepsin show that the enzyme is stored in oocytes as a 55 kDa zymogen. The activation of the zymogen occurs in vitro only at low pH. In vitro activation in the presence of cysteine protease inhibitors is blocked at an intermediary polypeptide of 48 kDa. Kinetic studies of this activation process at pH 3.5 and 4.6 show that the zymogen is processed in a manner similar to that of pepsin (Foltmann, 1986) and papain (Vernet et al., 1991). We propose that Musca domestica cathepsin zymogen activation occurs in two steps. First, an intramolecular cleavage of the procathepsin polypeptide chain (55,000), induced by low pH gives rise to an intermediary polypeptide (48,000) which then undergoes autolysis to produce the mature enzyme (41,000).     

Structural characterization of activation 'intermediate 2' on the pathway to human gastricsin

The crystal structure of an activation intermediate of human gastricsin has been determined at 2.4 A resolution. The human digestive enzyme gastricsin (pepsin C) is an aspartic proteinase that is synthesized as the inactive precursor (zymogen) progastricsin (pepsinogen C or hPGC). In the zymogen, a positively-charged N-terminal prosegment of 43 residues (Ala 1p-Leu 43p; the suffix 'p' refers to the prosegment) sterically prevents the approach of a substrate to the active site. Zymogen conversion occurs in an autocatalytic and stepwise fashion at low pH through the formation of intermediates. The structure of the non-covalent complex of a partially-cleaved peptide of the prosegment (Ala 1p-Phe 26p) with mature gastricsin (Ser 1-Ala 329) suggests an activation pathway that may be common to all gastric aspartic proteinases.     

The crystal structure of human procathepsin K

Cathepsin K is a cysteine protease present in human osteoclasts that plays an important role in bone resorption. Cathepsin K is synthesized as an inactive proenzyme and activated under conditions of low pH. Autoproteolytic processing of the N-terminal 99 amino acid propeptide produces the active, mature form of cathepsin K. It is presumed that the activation of procathepsin K in vivo occurs in the bone resorption pit, which has a low-pH environment. We have determined the structure of human procathepsin K at 2.8 A resolution. The structure of the mature enzyme domain within procathepsin K is virtually identical to that of mature cathepsin K. The fold of the propeptide of procathepsin K is similar to that observed in procathepsins B and L despite differences in length and sequence. A portion of the propeptide occupies the active site cleft of cathepsin K. Hydrophobic interactions, salt bridges, and hydrogen-bonding interactions are observed in the structure of the propeptide and between the propeptide and the mature enzyme of procathepsin K. These interactions suggest an explanation for the stability of the proenzyme. The structure of procathepsin K contributes to an understanding of the molecular basis of inhibition by the propeptide portion of the molecule and activation of this important member of the cysteine protease family.     

The propeptide domain of membrane type 1 matrix metalloproteinase is required for binding of tissue inhibitor of metalloproteinases and for activation of pro-gelatinase A

Activation of secreted latent matrix metalloproteinases (MMPs) is accompanied by cleavage of the N-terminal propeptide, thereby liberating the active zinc from binding to the conserved cysteine in the pro-domain. It has been assumed that an analogous mechanism is responsible for the activation of membrane type 1 MMP (MT1-MMP). Using recombinant wild-type MT1-MMP cDNA and mutant cDNAs transfected into COS-1 cells lacking endogenous MT1-MMP, we have examined the function of the propeptide domain of MT1-MMP. MT1-MMP was characterized by immunoblotting, surface biotinylation, gelatin substrate zymography, and 125I-tissue inhibitor of metalloproteinases 2 (TIMP-2) binding. In contrast to wild-type MT1-MMP-transfected COS-1 cells, transfected COS-1 cells containing a deletion of the N-terminal propeptide domain of MT1-MMP or a chimeric construction (substitution of the pro-domain of MT1-MMP with that of collagenase 3) were functionally inactive in terms of binding of 125I-labeled TIMP-2 to the cell surface and initiating the activation of pro-gelatinase A. These results support the concept that in its native plasma membrane-inserted form, the pro-domain of MT1-MMP plays an essential role in TIMP-2 binding and subsequent activation of pro-gelatinase A.     

Influence of cofactor binding and active site occupancy on the conformation of the macromolecular substrate exosite of factor VIIa

The catalytic activity of the trypsin-like serine protease coagulation factor VIIa is allosterically regulated. In this work, we employed monoclonal antibodies as probes to analyze conformational changes in the VII protease domain that are induced by zymogen activation, cofactor tissue factor (TF) binding, and active site occupancy. The epitopes of three monoclonal antibodies were mapped using a panel of 57 individual alanine replacement mutants in the protease domain. Two of the antibodies had typical "hot spot" epitopes in a basic cluster above the active site cleft and antibody binding to these epitopes was not affected by zymogen activation, TF binding, or active site occupancy. In contrast, the binding kinetics of VII/VIIa to a monoclonal antibody that mapped to an extended epitope overlapping with the macromolecular substrate exosite was affected by each of the conformational transitions of the VIIa protease domain. The changes in antibody affinity are consistent with a transition from zymogen VII to the TF.VIIa complex, with free enzyme VIIa as an intermediate that retains some zymogen-like features responsible for its low catalytic activity. In contrast, active site occupancy resulted in effects that were qualitatively different from the effects of zymogen activation on the antibody epitope. This provides novel insight into the conformational interdependence between the active site, the region for macromolecular substrate recognition, and the cofactor binding exosite of this allosterically regulated serine protease.     

Reciprocated matrix metalloproteinase activation: a process performed by interstitial collagenase and progelatinase A

Gelatinase A, a member of the matrix metalloproteinase (MMP) family, is secreted possessing an 80 amino acid N-terminal propeptide that must be removed in order to generate the active enzyme. Purified progelatinase A was activated to 38% of maximum by a 6 h incubation at 37 degrees C with equimolar concentrations of trypsin-activated interstitial collagenase (another MMP). The increase in activity was accompanied by cleavage of the M(r) 72,000 progelatinase A to the M(r) 66,000 active enzyme that has Y81 as its N-terminus. At low concentrations, progelatinase A was processed via an inactive intermediate, suggesting that its activation is a biphasic process. This was confirmed by the action of collagenase on proE375-->A (a mutant of progelatinase A that cannot become active) because, in this instance, only an M(r) 68,000 species with L38 as the N-terminus was produced. The remaining propeptide amino acids to Y81 could be readily removed by added active gelatinase A, indicating that collagenase works by generating an intermediate that is susceptible to autolytic activation. Although relatively slow, the rate of activation could be increased approximately 10-fold by the addition of 100 micrograms/mL heparin. This binds to the C-terminal domain of collagenase and progelatinase A and presumably acts as a template that positions the reactants close to one another. Collagenase activated by trypsin retains 8 or 14 amino acids of its propeptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyuria and polydipsia in the diagnosis of endocrine diseases

Elevated plasma von Willebrand factor (vWf) levels are found in diabetes and other vasculopathies, and predict cardiovascular mortality. vWf is stored and released from endothelial cell secretory granules, along with equimolar amounts of its propeptide (vWf:AgII). In the present study, we examined plasma propeptide levels as a marker of endothelial secretion in vivo, using an ELISA based on monoclonal antibodies. vWf but not propeptide levels are influenced by blood groups, explaining in part the smaller variation in plasma propeptide levels among normal individuals. In both controls and insulin-dependent diabetic patients, we found a close correlation between propeptide and immunoreactive vWf levels (r2=0.54, p <0.0001). vWf and propeptide were elevated in patient subgroups with microalbuminuria or overt diabetic nephropathy, whereas only the propeptide was significantly elevated in the normoalbuminuric subgroup. This observation suggests that in conjunction with vWf, propeptide measurements may improve the identification of endothelial activation, which occurs frequently even without increased urinary albumin excretion. In 12 NIDDM patients, a 3-week diet enriched in monounsaturated fat (MUFA) resulted in parallel decreases in vWf (-22%, p <0.05) and propeptide (-17%, p <0.05) levels, indicating that the experimental diet affected endothelial secretion rather than vWf catabolism. A carbohydrate-enriched control diet did not significantly influence either marker. Our results suggest that concomitant determinations of plasma vWf and propeptide are useful tools to assess endothelial activation in vivo, and reinforce our previous conclusion that a diet rich in MUFA can improve endothelial function in NIDDM.     

Activating a zymogen without proteolytic processing: mutation of Lys15 and Asn194 activates trypsinogen

The zymogen and mature enzyme forms of trypsin-like serine proteases exhibit a wide range of activities. The prototypical trypsinogen-trypsin system is an example of a minimally active zymogen and a maximally active mature protease. The present work identifies several features of trypsinogen which govern its activity. Our results indicate that rat trypsin is 10(8)-fold more active than rat trypsinogen. Rat trypsinogen appears to be less active than bovine trypsinogen. His40 is believed to be an important determinant of zymogen activity. We are unable to verify this role for His40 in trypsinogen since the mutation of His40 to Phe appears to change the trypsin-substrate interface. Deletion of the N-terminal Ile16 from trypsin is expected to produce a trypsinogen-like protein since the Ile16-Asp194 salt bridge cannot form. Such mutants have higher activity and BPTI affinity than trypsinogen, which indicates that the activation peptide stabilizes the inactive trypsinogen conformation. The mutation of Lys15 to Ala increases the BPTI affinity and activity of trypsinogen to an even greater extent; thus, removal of Lys15 can account for the effect of the loss of the activation peptide. These results suggest that Lys15 is an important determinant of zymogen activity. The mutation of Asp194 to Asn also increases the BPTI affinity and activity of trypsinogen. This result suggests that in addition to stabilizing the active conformation of trypsin via the Ile16-Asp194 salt bridge, Asp194 also maintains the inactive conformation of trypsinogen. A correlation exists between the values of kcat/Km and BPTI affinity of mutant trypsinogens and trypsins. However, the slope of this correlation is 0.64, which indicates that different "active" conformations are involved in BPTI binding and substrate hydrolysis. DeltaI16V17 trypsinogen is the lone outlier; its BPTI affinity is higher than would be expected based on the value of kcat/Km. We show that the rate of BPTI association is slower for DeltaI16V17 trypsinogen than for a mutant trypsinogen with a similar BPTI affinity. This observation suggests that BPTI binds to an "active" trypsinogen conformation that is not kinetically accessible to substrates.