The two human trypsinogens. Evidence of complex formation with basic pancreatic trypsin inhibitor-proteolytic activity.

The formation of complexes between human trypsinogens and the basic pancreatic trypsin inhibitor is demonstrated by using affinity chromatography on Sepharose coupled to basic pancreatic trypsin inhibitor. This interaction indicates the pre-existence of the active site in human trypsinogens. This active site induces the proteolytic activity of the two zymogens which activate spontaneously at pH 5.6 and pH 8.0 before and after affinity chromatography. The effect of affinity-chromatography on trypsinogen spontaneous activation is not the same on trypsinogens 1 and 2. A striking difference appears between the activation of the two trypsinogens. In all cases, trypsinogen 1 autoactivates more rapidly than trypsinogen 2, except at pH 5.6 in the presence of 10 mM Ca2+, which inhibits the autoactivation of trypsinogen 1. The effect of inherent proteolytic activity of human trypsinogens is discussed in relation to pathological conditions of enterokinase deficiency and acute pancreatitis.     

The inactive subunit of ruminant procarboxypeptidase A-S6 complexes. Structural basis of inactivity and physiological role

Subunit III has so far been found only in the pancreas of ruminants in a non-covalent association (procarboxypeptidase A-S6) with two different proteins: the procarboxypeptidase A itself (subunit I) and a C-type chymotrypsinogen (subunit II). In contrast with these latter two proteins, which are zymogens of pancreatic proteases, subunit III seems to be devoid of any activity towards specific substrates of pancreatic proteases. However, it possesses a weakly functional active site which allows it to hydrolyze a non-specific ester, p-nitrophenyl acetate, and to react with several active-site titrants. The binding of proflavin to subunit III shows that this protein owns a non-polar binding site with a very high Kd compared to that of chymotrypsin. The comparison of the amino acid sequences of subunit III and some serine proteases showed that subunit III is closely related to an elastase. Models of the tertiary structure of subunit III suggest a conformational modification that affects the substrate binding and could explain the lack of specific enzymatic activity. The presence of subunit III in the ternary complex is not related to an enzymatic function. This protein does not participate in the activation process of subunit I but prevents the denaturation of this subunit at low pH. This may represent its biological role in the acidic environment of the duodenum in ruminants.     

Comparative studies on the mechanism of activation of the two human trypsinogens

The activation of human trypsinogens 1 and 2 by porcine enterokinase at pH 5.6 shows that the two human zymogens are equivalent substrates for this enzyme and that both proteins are activated faster than the cationic bovine trypsinogen. At pH 8.0 and in the presence of 20 mM calcium the two human trypsinogens are activated by either human trypsin at the same rate but the affinity of both trypsins is higher for trypsinogen 1 than for trypsinogen 2. Two Ca2+ binding sites are identified in the two human zymogens and their pK(Ca2+) values determined. For trypsinogen 1 the values are respectively of 2.8 and 3.3 for the primary and secondary Ca2+ binding sites, and for trypsinogen 2 of 3.4 and 2.7. These values are markedly different from those obtained for bovine cationic trypsinogen, especially in the case of trypsinogen 1. These results point out a different degree of saturation of the calcium binding sites of the 2 human zymogens that must exist in physiological conditions, suggesting different biological activities of the two trypsinogens.     

Application of pulse radiolysis to the study of proteins: chymotrypsin and trypsin.

The one-electron reduction of chymotrypsin, trypsin, and their zymogens have been studied by pulse radiolysis. The optical spectra of the transient products from the two active enzymes display a pH-dependent band at 360 nm, associated with the histidine-electron adduct. The yield of the histidyl radical as a function of pH is consistent with a pK(a) less than 4.5, which suggests that the radical is located at the enzyme active site. The histidines of the proenzymes chymotrypsinogen and trypsinogen are unreactive towards the hydrated electron. We conclude that formation of the histidine-electron adduct at the serine protease active site is sensitive to the physical alterations which accompany protease activation.     

Hereditary pancreatitis-associated mutation asn(21) --> ile stabilizes rat trypsinogen in vitro.

Mutations Arg(117) --> His and Asn(21) --> Ile in human trypsinogen-I have been recently associated with hereditary pancreatitis (HP). The Arg(117) --> His substitution is believed to cause pancreatitis by stabilizing trypsin against autolytic degradation, while the mechanism of action of Asn(21) --> Ile has been unknown. In an effort to understand the effect(s) of this mutation, Thr(21) in the highly homologous rat trypsinogen-II was replaced with Asn or Ile, and the recombinant zymogens and their active trypsin forms were studied. Kinetic parameters of all three trypsins were comparable, and the active enzymes suffered autolysis at similar rates, indicating that neither catalytic properties nor proteolytic stability of trypsin are influenced by mutations at position 21. When incubated at pH 8.0, 37 degrees C, pure zymogens underwent autoactivation with concomitant trypsinolytic degradation in a Ca(2+)-dependent fashion. Thus, in the presence of 5 mM Ca(2+), autoactivation and digestion of the zymogens after Arg(117) and Lys(188) were observed, while in the presence of 1 mM EDTA autoactivation and cleavage at Lys(188) were reduced, and zymogenolysis at the Arg(117) site was enhanced. Overall rates of zymogen degradation in [Asn(21)]- and [Ile(21)]trypsinogens were higher in Ca(2+) than in EDTA, while [Thr(21)]trypsinogen demonstrated inverse characteristics. Remarkably, both in the presence and absence of Ca(2+), [Ile(21)]trypsinogen exhibited significantly higher stability against autoactivation and proteolysis than zymogens with Asn(21) or Thr(21). The observations suggest that autocatalytic trypsinogen degradation may be an important defense mechanism against excessive trypsin generation in the pancreas, and trypsinogen stabilization by the Asn(21) --> Ile mutation plays a role in the pathogenesis of HP.     

An inexpensive device for sample stirring in VSU Zeiss spectrophotometers. Its use for kinetic measurements and active site titrations with immobilized enzymes.

The construction of a simple and effective sample stirring device for commercial spectrophotometers and its use for continuous kinetic measurements and active site titrations with immobilized enzymes is described. Sepharose-bound leucine aminopeptidase and trypsin were selected as model enzymes to test the performance of the magnetic stirring equipment. Kinetic parameters of insolubilized leucine aminopeptidase using L-leucine p-nitroanilide as substrate and the catalytic site concentration of matris-bound trypsin using p-nitrophenyl p'-guanidinobenzoate as active site titrant could be evaluated without significant interference from the turbidity of the stirred Sepharose suspension. The problem of grinding of the support material could be overcome. Both unbound native and carrier-fixed enzyme may be reacted under identical conditions with similar convenience and sensitivity.     

Double-headed protease inhibitors from black-eyed peas. VI. Singlet-singlet energy transfer and other optical studies on the structure of trypsin and chymotrypsin complexes.

The geometry of the binary and ternary complexes of two black-eyed pea inhibitors with trypsin and chymotrypsin has been established by distance measurements using the technique of singlet-singlet energy transfer. Triangulation of measured distances in the ternary double-headed complex of the trypsin-chymotrypsin inhibitor (BEPCI) with trypsin and chymotrypsin limits the possible structural models for this complex to those in which the center to center distance between trypsin and chymotrypsin is about 64 A, the distance from the center of trypsin to the single fluorescently labeled tyrosyl residue of the BEPCI dimer is about 33 A, and the distance between the chymotrypsin center and the labeled tyrosine of the inhibitor is about 43 A. Energy transfer results for the trypsin inhibitor (BEPTI) complexes show conclusively that the weak trypsin site is structurally analogous to the strong chymotrypsin binding site of BEPCI. The weak chymotrypsin binding site of BEPTI is structurally analogous to the strong trypsin sites of BEPCI and BEPTI. Corresponding distances in binary and ternary complexes are the same, indicating that little or no structural rearrangement occurs when the ternary complexes are formed. Complex formation was shown to involve tryptophan and tryosine residues of both trypsin and chymotrypsin as judged by absorption and circular dichroism difference spectroscopy. In addition, circular dichroism difference spectra revealed some disulfide contributions.     

Thyroliberin stimulates rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate by a phosphodiesterase in rat mammotropic pituitary cells. Evidence for an early Ca2+-independent action.

Benzyl p-guanidinothiobenzoate hydrochloride was synthesized and demonstrated to be useful for active-site titration of bovine trypsin, bovine thrombin, human lung tryptase, bovine activated protein C, human Factor XIIa fragment and bovine Factor Xa beta. The titration is based on rapid formation of a stable acyl-enzyme with a stoichiometric release of benzyl thiol. Thiol production is measured quantitatively by including 4,4'-dithiodipyridine in the reaction mixture and measuring the increase in absorbance at 324 nm. Ellman's reagent has also been successfully employed, allowing measurement at 410 nm. Unlike p-nitrophenyl p'-guanidinobenzoate, the thioester titrant reacts slowly with chymotrypsin A alpha thus eliminating interference by this enzyme in most titrations. Advantages of this reagent as a titrant include: flexibility in detection of the released thiol, selectivity between trypsin and chymotrypsin-like enzymes, minimal pH-dependence of the epsilon of the absorbing species, relative stability of the reagent under titration conditions, and high epsilon at pH 7.2 with either 4,4'-dithiodipyridine or Ellman's reagent. The reagent should prove useful as an alternative to p-nitrophenyl p'-guanidinobenzoate hydrochloride for the determination of active-site concentrations of the enzymes employed, as well as of other related enzymes.     

Dynamic and structural properties of the calcium binding site of bovine serine proteases and their zymogens. A multinuclear nuclear magnetic resonance and stopped-flow study

The combined use of 43Ca and 113Cd nuclear magnetic resonance (n.m.r.) has provided information on the structural and dynamic properties of the calcium binding site located in homologous positions in bovine beta-trypsin, alpha-chymotrypsin and their zymogens. The 43Ca and 113Cd n.m.r. chemical shifts are consistent with an octahedral symmetry of the binding site and with the substitution of one of the two carboxylate ligands present in trypsin(ogen) with a neutral ligand in chymotrypsin(ogen). The constancy of the 113Cd n.m.r. chemical shifts upon binding of the pancreatic trypsin inhibitor and/or the dipeptide Ile-Val to trypsinogen confirms that structural changes in the activation domain do not affect the calcium binding site. The exchange between bound and "free" (solvated) Ca2+ is slow on the 43Ca n.m.r. time-scale for trypsin(ogen), but falls in the intermediate exchange region for chymotrypsin(ogen). In trypsin, the Ca2+ off-rate was measured by stopped-flow making use of the calcium indicator 1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid and was found to be 3(+/- 1) s-1. In chymotrypsin(ogen) the off-rates calculated from the 43Ca n.m.r. data are 70 s-1 and 350 s-1, respectively. The dynamic properties of the calcium binding site of serine (pro)enzymes have been related to the flexibility of the binding site itself and have been compared to those of other extracellular and intracellular calcium binding proteins.     

Comparison of anionic and cationic trypsinogens: the anionic activation domain is more flexible in solution and differs in its mode of BPTI binding in the crystal structure

Unlike bovine cationic trypsin, rat anionic trypsin retains activity at high pH. This alkaline stability has been attributed to stabilization of the salt bridge between the N-terminal Ile16 and Asp194 by the surface negative charge (Soman K, Yang A-S, Honig B, Fletterick R., 1989, Biochemistry 28:9918-9926). The formation of this salt bridge controls the conformation of the activation domain in trypsin. In this work we probe the structure of rat trypsinogen to determine the effects of the surface negative charge on the activation domain in the absence of the Ile16-Asp194 salt bridge. We determined the crystal structures of the rat trypsin-BPTI complex and the rat trypsinogen-BPTI complex at 1.8 and 2.2 A, respectively. The BPTI complex of rat trypsinogen resembles that of rat trypsin. Surprisingly, the side chain of Ile16 is found in a similar position in both the rat trypsin and trypsinogen complexes, although it is not the N-terminal residue and cannot form the salt bridge in trypsinogen. The resulting position of the activation peptide alters the conformation of the adjacent autolysis loop (residues 142-153). While bovine trypsinogen and trypsin have similar CD spectra, the CD spectrum of rat trypsinogen has only 60% of the intensity of rat trypsin. This lower intensity most likely results from increased flexibility around two conserved tryptophans, which are adjacent to the activation domain. The NMR spectrum of rat trypsinogen contains high field methyl signals as observed in bovine trypsinogen. It is concluded that the activation domain of rat trypsinogen is more flexible than that of bovine trypsinogen, but does not extend further into the protein core.     

Effect of B-ring substituents on absorption and circular dichroic spectra of colchicine analogues.

Near-ultraviolet absorption and circular dichroic spectra of several B-ring derivatives of colchicine have been obtained in a variety of solvents. The spectra of the molecules in solvent were analyzed and compared with spectra of the molecules bound to tubulin. Absorption spectra of deacetamidocolchicine, deacetylcolchicine, demecolcine, and N-methyldemecolcine [B-ring substituents = H, NH2, NHCH3, and N(CH3)2, respectively] were analyzed by multiple differentiation of the spectrum. It was found that an amine substituent at the C-7 position on the B-ring of the colchicinoid affected the higher energy transition of the near-ultraviolet spectra of the colchicinoid in the absence of tubulin in a manner consistent with a hyperconjugative alteration of this transition. The fourth derivatives of the absorption spectra of all four molecules bound to tubulin were similar to each other and to colchicine. As was true in the case of colchicine, the negative near-ultraviolet circular dichroic band of the aminoclochicinoids was relatively unaffected by solvent, but the molar ellipticity of the band was greatly reduced with tubulin binding. It is concluded that the binding site environments of the B-ring analogues of colchicine, as probed by absorption and circular dichroic spectroscopy, are equivalent.     

Demonstration of human pancreatic anionic trypsinogen in normal serum by radioimmunoassay

A specific radioimmunoassay for human pancreatic anionic trypsin has been developed. The trypsin employed as radioiodinated tracer in the assay was inactivated with tosyl-L-lysine chloromethyl ketone in order to prevent binding of the tracer to the serum inhibitors α₁-antitrypsin and α₂-macroglobulin. A normal serum level of immunoreactive anionic trypsin of 5.45 ng/ml was determined. The results of experiments in which serum was fractionated by Sephadex G-200 gel filtration suggest that essentially all of the immunoreactive material in normal human serum is trypsinogen. This finding implies that a small fraction of the zymogens synthesized in the pancreas are released directly into the circulation.     

Human mesotrypsin is a unique digestive protease specialized for the degradation of trypsin inhibitors

Mesotrypsin is an enigmatic minor human trypsin isoform, which has been recognized for its peculiar resistance to natural trypsin inhibitors such as soybean trypsin inhibitor (SBTI) or human pancreatic secretory trypsin inhibitor (SPINK1). In search of a biological function, two conflicting theories proposed that due to its inhibitor-resistant activity mesotrypsin could prematurely activate or degrade pancreatic zymogens and thus play a pathogenic or protective role in human pancreatitis. In the present study we ruled out both theories by demonstrating that mesotrypsin was grossly defective not only in inhibitor binding, but also in the activation or degradation of pancreatic zymogens. We found that the restricted ability of mesotrypsin to bind inhibitors or to hydrolyze protein substrates was solely due to a single evolutionary mutation, which changed the serine-protease signature glycine 198 residue to arginine. Remarkably, the same mutation endowed mesotrypsin with a novel and unique function: mesotrypsin rapidly hydrolyzed the reactive-site peptide bond of the Kunitz-type trypsin inhibitor SBTI, and irreversibly degraded the Kazal-type temporary inhibitor SPINK1. The observations suggest that the biological function of human mesotrypsin is digestive degradation of trypsin inhibitors. This mechanism can facilitate the digestion of foods rich in natural trypsin inhibitors. Furthermore, the findings raise the possibility that inappropriate activation of mesotrypsinogen in the pancreas might lower protective SPINK1 levels and contribute to the development of human pancreatitis. In this regard, it is noteworthy that the well known pathological trypsinogen activator cathepsin B exhibited a preference for the activation of mesotrypsinogen of all three human trypsinogen isoforms, suggesting a biochemical mechanism for mesotrypsinogen activation in pancreatic acinar cells.     

Thioltrypsin. Chemical transformation of the active-site serine residue of Streptomyces griseus trypsin to a cysteine residue.

The active-site serine residue of Streptomyces griseus trypsin was converted to a cysteine residue, and the product, thioltrypsin, was purified through two chromatographic steps with organomercurial-Sepharose and soybean trypsin inhibitor-Sepharose as specific adsorbents. The purified preparation of thioltrypsin was found to contain a single residue of cysteine and to react with almost equimolar amounts of normality titrants. It exhibited only traces of catalytic activity toward typical trypsin substrates such as Nalpha-tosyl-L-arginine methyl ester, whereas it retained some activity toward "active ester" substrates such as Nalpha-carbobenzoxy-L-lysine p-nitrophenyl ester. The activity was inhibited by sulfhydryl-blocking reagents, but no inhibition was observed by reagents reactive with the active hydroxyl group of serine proteases. Leupeptin, a natural trypsin inhibitor of peptidyl nature, also inhibited thioltrypsin. Some difference in the mode of leupeptin inhibition, however, was detected between trypsin and thioltrypsin. The bindings of small synthetic ligands and soybean trypsin inhibitor to thioltrypsin were compared with those to trypsin.     

Synthetic inhibitors of trypsin, plasmin, kallikrein, thrombin, C1r-, and C1 esterase.

p-Carbethoxyphenyl episol-guanidinocaproate and p-(p'-guanidinobenzoyloxy)-phenyl derivatives were prepared, and their inhibitory effects on trypsin, plasmin, plasma kallikrein, thrombin, C1r- and C1 esterase were examined. Among the various inhibitors tested, p-nitrophenyl p'-guanidinobenzoate, N,N-dimethylamino p-(p'-guanidinobenzoyloxy)-benzoyl glycolate and N,N-dimethylamino p-(p'-guanidinobenzoyloxy)-benzilcarbonyloxy glycolate were the most effective inhibitors of trypsin, plasmin, plasma kallikrien and thrombin, and they strongly inhibited the esterolytic activities of C1r- and C1 esterase.     

Compatible solutes as protectants for zymogens against proteolysis

Compatible solutes are small organic osmoprotectants that have the capability to stabilize proteins. In coupled assays, the effect of the solutes ectoine, hydroxyectoine and betaine on the activation of the zymogens trypsinogen and chymotrypsinogen, catalyzed by enteropeptidase and trypsin, respectively, was studied. To different extents, all solutes protected the zymogens against activation. Ectoine (800 mM) was the most potent solute in reducing the formation of trypsin to 4% of the control value and of chymotrypsin to 23%. In separate experiments, the ability of the solutes to preserve proteolytic activity during incubation was investigated. After 4 h, trypsin and chymotrypsin completely lost their activity, but in the presence of ectoine, approximately 50% residual activity was maintained. It is proposed that a conformational shift of the protein towards folded, native-like states induced by preferential exclusion of the solute is responsible for the stabilizing and chaperone-like effects.     

The identification of neotrypsinogens in samples of bovine trypsinogen

Isoelectric focusing of commercial samples of bovine trypsinogen detected a component with a lower isoelectric pH than that of trypsinogen. The isoelectric pH was 8.75 compared to 9.3 for trypsinogen, and the amount of the component varied from 16 to 41% of the total protein. The protein (24,000 Da) was converted to fragments of 13,800 and 10,500 Da on reduction with dithioerythritol, showing that the component was a modified form of trypsinogen containing a cleaved peptide bond. The cleavage site was established from the study of four polypeptide fragments which were isolated from the fully reduced and S-carboxymethylated trypsinogen. The molecular weights, amino acid compositions, and amino-terminal sequences of these fragments identified a cleavage of Lys 131-Ser 132, namely from a Ser-neotrypsinogen, or at Arg 105-Val 106, from a Val-neotrypsinogen. Val-neotrypsinogen was the more abundant of the two and was approximately 71% of the total neotrypsinogen in the trypsinogen sample. Both neotrypsinogens were converted to active trypsin molecules in high yields, showing that the zymogens closely resembled the conformation of intact trypsinogen. Presumably, the neotrypsinogens were produced during the isolation of the zymogen when pancreatic tissue was partly autolyzed and active trypsin was present.     

Comparative in vitro studies on native and recombinant human cationic trypsins. Cathepsin B is a possible pathological activator of trypsinogen in pancreatitis

Hereditary pancreatitis, an autosomal dominant disease is believed to be caused by mutation in the human trypsinogen gene. The role of mutations has been investigated by in vitro studies using recombinant rat and human trypsinogen (TG). In this study we compare the enzymatic properties and inhibition by human pancreatic secretory trypsin inhibitor (hPSTI) of the native, postsynthetically modified and recombinant cationic trypsin, and found these values practically identical. We also determined the autolytic stability of recombinant wild type (Hu1Asn21) and pancreatitis-associated (Hu1Ile21) trypsin. Both forms were equally stable. Similarly, we found no difference in the rate of activation of the two zymogens by human cationic and anionic trypsin. Mesotrypsin did not activate either form. The rate of autocatalytic activation of Hu1Asn21 TG and Hu1Ile21 TG was also identical at pH 8 both in the presence and absence of Ca2+. At pH 5 Hu1Ile21 TG autoactivated about twice as fast as Hu1Asn21 TG. The presence of physiological amount of hPSTI completely prevented autoactivation of both zymogens at pH 8 and at pH 5 as well. Cathepsin B readily activated both zymogens although Hu1Ile21 TG was activated about 2.5-3 times as fast as Hu1Asn21 TG. The presence of hPSTI did not prevent the activation of zymogens by cathepsin B. Our results underlie the central role of cathepsin B in the development of different forms of pancreatitis.     

Functional significance of flexibility in proteins

The structural basis and the functional implications of large-scale flexibility are discussed for three systems: trypsin–trypsinogen, immunoglobulins, and citrate synthase. The trypsin–trypsinogen system provides an example in which an order–disorder transition is used as a means to regulate enzymatic activity. Immunoglobulins demonstrate how flexibly linked domains may be used to allow the binding of ligands with diverse arrangements. In citrate synthase, domain motion forms an active site that is shielded from solvent. Analogous large-scale flexibility has been observed in a number of other systems.     

Circular dichroism of soybean leghemoglobin.

Circular dichroic (CD) spectra of soybean leghemoglobin, and some of its liganded derivatives were measured over the wavelength range of 650 to 200 nm. The heme-related circular dichroic bands in the visible, Soret and ultraviolet wavelength regions exhibit Cotton effects characteristic of each of the compounds examined. The positions of the dichroic bands vary with ligand substitutions and the oxidation state of the iron. All leghemoglobin derivatives, except the apoprotein, exhibit negative circular dichroic bands in the region of Soret absorption. In this region the optical activity of compounds with high-spin moments is greater than that of compounds with low or intermediate spin moments. The ellipticity of the heme band at about 260 nm is also altered by ligand binding and spin state. The dichroic spectra in the far-ultraviolet region indicated a high extent of alpha-helical structure (about 70%) in the native leghemoglobin and its liganded derivatives. The helicality of the apoprotein seems to diminish suggesting a decrease caused by the removal of the heme.