Kinetic behaviour of proenzymes activation in the presence of different inhibitors for both activating and activated enzymes

In the present paper, a kinetic analysis of a general model for proenzyme activation, where the activating enzyme and also the activated one are reversibly inhibited in two steps by two different inhibitors, has been performed. The cases in which both inhibitors are the same, or in which the inhibition is irreversible (only one or the two inhibition routes) are treated as particular cases of the general model. In addition, the kinetic behaviour of many other proenzyme activation systems involving inhibition, particular cases of the reaction scheme under study, can be obtained. The total number of particular cases for the general model under study is 370, so this approach offers to the scientific community working in limited proteolysis regulation for the first time a method based on general solutions which only needs to be specified to their concrete problem of zymogen activation. Finally, new adimensional parameters are introduced, allowing the knowledgement, in the case that any of the inhibition routes is irreversible, the relative weight of both activation and irreversible inhibition routes.     

Structure and function of microplasminogen: II. Determinants of activation by urokinase and by the bacterial activator streptokinase.

We have used a group of human microplasminogens (mPlg), modified by residue substitutions, insertions, deletions, and chain breaks (1) to study the determinants of productive interactions with two plasminogen activators, urokinase (uPA), and streptokinase (SK); (2) to explore the basis of species specificity in the zymogen-SK complex activity; and (3) to compare active SK complex formation in mPlg and microplasmin (mPlm). Modifications within the disulfide-bonded loop containing the activation site and the adjacent hexadecapeptide upstream sequence showed that uPA recognition elements encompassed R29 at the activation site and multiple elements extending upstream to perhaps 13 residues, all maintained in specific conformational register by surrounding pairs of disulfide bonds. A generally parallel pattern of structural requirements was observed for active zymogen-SK complex formation. Changes within the loop downstream of the activation site were tolerated well by uPA and poorly by SK. The introduction of selected short bovine (Plg) sequences in human mPlg reduced the activity of the resulting SK complexes. The requirements for active SK complex formation are different for mPlg and mPlm.     

Plasminogen-binding centers of molecules of fibrinogen, fibrin and products of their proteolysis

Using affinity chromatography, the binding of Lys-plasminogen to fibrinogen, fibrin and the consecutively formed products of their proteolysis was studied. The optimal conditions for this binding were elaborated, and the quantitative parameters of Lys-plasminogen binding to fibrinogen-Sepharose were determined. It was found that the interaction of Lys-plasminogen with fibrinogen- and fibrin-Sepharose is provided for by the lysine-binding sites of the proenzyme molecule. After partial hydrolysis of fibrinogen by plasmin, the amount of adsorbed plasminogen increases and the type of binding changes; part of the proenzyme molecules bind in the presence of 0.003 M 6-aminohexanoic acid, i.e., when lysine-binding sites appear to be blocked. A comparative study of plasminogen binding to fibrinogen fragments was carried out. The resistance of the complexes formed to the effect of 6-aminohexanoic acid and arginine competing for the binding sites was determined. The data obtained testify to the appearance of additional plasminogen-binding sites in the fibrinogen molecule during proteolysis. These sites are complementary for both lysine-and arginine-binding sites of the plasminogen molecule and are localized in the peripheral domains of the fibrinogen molecule.     

Comparison of calcium-activated, cyclic nucleotide-independent protein kinase and adenosine 3':5'-monophosphate-dependent protein kinase as regards the ability to stimulate glycogen breakdown in vitro.

A cyclic nucleotide-independent protein kinase, which was produced from its proenzyme upon limited proteolysis by a Ca2+-dependent protease (Takai, Y., Yamamoto, M., Inoue, M., Kishimoto, A., & Nishizuka , Y. (1977) Biochem. Biophys. Res. Commun. 77, 542-550), showed an ability to phosphorylate not only muscle glycogen phosphorylase kinase but also glycogen synthase, resulting in activation and inactivation of the respective enzymes, although the protein kinase was less active than adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase toward glycogen synthase. Available evidence indicates that this new protein kinase shows pleiotropic functions apparently similar to those described for cyclic AMP-dependent protein kinase. Nevertheless, these protein kinases were clearly distinguishable from each other in their response to cyclic nucleotides and susceptibility to protein inhibitor.     

Bovine proacrosin from cauda epididymal sperm: purification, characterization and partial sequencing at N-terminus.

1. In the present study, we isolated the two forms of proacrosin from acid extracts (pH 3.0) of cauda epididymal bovine spermatozoa by ammonium sulfate fractionation, gel filtration on Sephadex G-150 and affinity chromatography on Concanavalin A Sepharose 4B. The overall purification was 13-fold with respect to crude acid acrosomal extract. 2. The apparent molecular weight of the proacrosins determined by SDS-PAGE were 44,000 and 38,000. Both forms have proteinase activity on gelatin-SDS-polyacrylamide gel electrophoretic zymography. 3. The M(r) = 38,000 component was isolated by reverse phase HPLC. Thirty-nine amino acid residues at the N-terminus have about 72 and 77% sequence similarity with boar and human proacrosin, respectively. 4. The amino acid sequence of 14 amino acids at the N-terminus of the high molecular weight component (M(r) = 44,000) was determined after electroblotting on a polyvinylidene difluoride membrane. This portion of the molecule is identical with that of the low molecular weight component. 5. Proacrosin autoactivation followed the sigmoidal activation curve.     

Zymogen activation: effect of peptides sequentially related to the bovine beta-trypsin N-terminus on Kazal inhibitor and benzamidine binding to bovine trypsinogen

The activating effect of peptides sequentially related to the Ile 16-Val17-Gly18 N-terminus of bovine beta-trypsin (namely Ile-Val-Gly, Ile-Val, Ile-Leu, Ile-Ala, Val-Val, Leu-Val, and Val-Leu) on the thermodynamic parameters for the binding of the porcine pancreatic secretory trypsin inhibitor (Kazal inhibitor) and benzamidine to bovine trypsinogen was investigated at pH 5.5 (Bis tris-HCl buffer, I = 0.1 M) and T = 21 +/- 0.5 degrees C. Thermodynamic parameters for Kazal inhibitor and benzamidine association to the binary peptide/zymogen adducts are more favorable than those observed for ligand binding to the proenzyme alone, although never as much as those reported for the formation of bovine beta-trypsin/Kazal inhibitor and bovine beta-trypsin/benzamidine adducts. Analogously, the affinity of activating peptides for the binary proenzyme/Kazal inhibitor and binary proenzyme/benzamidine complexes is higher than that observed for peptide binding to free bovine trypsinogen. Differences in affinity for ligand binding to free bovine trypsinogen, to its binary adducts and to bovine beta-trypsin suggest the presence of different activation levels of the proenzyme, none of which structurally coincide with that achieved in bovine beta-trypsin. The existence of different discrete states suggests that the zymogen-to-active enzyme transition should not be considered as a two-state process but as a multistep event.     

Human progastricsin. Analysis of intermediates during activation into gastricsin and determination of the amino acid sequence of the propart

Human progastricsin was prepared from extracts of gastric mucosa by chromatography on columns of DEAE-cellulose. The amino acid compositions of progastricsin and gastricsin were determined and calculated on the basis of the molecular weights 38 000 and 32 000 respectively. The activation of progastricsin at pH 2 was investigated and monitored by agarose gel electrophoresis at pH 5.4. Two intermediates were observed. Determination of the amino acid sequence showed that the propart consists of 43 amino acid residues. A pronounced homology with other gastric zymogens was found. With the proenzyme amino acid residue numbering used previously [B. Foltman (1981) Essays in Biochemistry, 17, 52-84] the activation of progastricsin at pH 2 may be summarized as follows. The first cleavage occurs after Phe (p27). At pH 5.4 the peptide remains associated with the protein (intermediate I). Subsequent proteolysis removes the peptides from Leu (p28) to Leu (p45). At pH 5.4 the N-terminal peptide from progastricsin (p2-p27) remains associated with gastricsin (intermediate II) until the propart peptide is hydrolysed to smaller fragments.     

Cynomolgus Monkey (Macaca fascicularis) Cathepsin K: Cloning, Expression, Purification, and Activation

Methodology for the production of recombinant active cynomolgus monkey (Macaca fascicularis) cathepsin K (EC 3.4.22.38) was elucidated. The cDNA encoding the cathepsin K was cloned from femaleM. cynomolgusmonkey mRNA. The deduced amino acid sequence ofM. cynomolguspreprocathepsin K from the cDNA sequence showed 94.2% identity to human preprocathepsin K. Sequence differences occurred only in the prepro- domains; the mature domains were identical. The recombinantM. cynomolguscathepsin K was expressed as a secreted proenzyme using baculovirus-infected SF21 insect cells having the predicted N-terminus (LYPEEILDTH … ), indicating proper cleavage of the secretion sequence. Purified monkey procathepsin K was activated under autocatalytic conditions at pH 4.0. The mature enzyme was composed of mixture of enzymes having N-termini of Gly¹¹³and Arg¹¹⁴. The molecular weight was determined to be 23,668.3 Da by MALDI-TOF-MS which is consistent with the absence of carbohydrate on the mature enzyme. These results indicate that monkey procathepsin K is able to autoactivate and produces a mature enzyme which is identical to that of human cathepsin K. Since the sequence of monkey and human mature cathepsin K are identical and thein vitroactivation mechanisms appear to be indistinguishable, monkeys are predicted to be a good animal model for evaluating cathepsin K inhibitorsin vivoas therapeutic agents for diseases characterized by excessive bone loss, such as osteoporosis.     

The high-resolution crystal structure of porcine pepsinogen.

The structure of porcine pepsinogen at pH 6.1 has been refined to an R-factor of 0.173 for data extending to 1.65 A. The final model contains 180 solvent molecules and lacks density for residues 157-161. The structure of this aspartic proteinase zymogen possesses many of the characteristics of pepsin, the mature enzyme. The secondary structure of the zymogen consists predominantly of beta-sheet, with an approximate 2-fold axis of symmetry. The activation peptide packs into the active site cleft, and the N-terminus (1P-9P) occupies the position of the mature N-terminus (1-9). Thus changes upon activation include excision of the activation peptide and proper relocation of the mature N-terminus. The activation peptide or residues of the displaced mature N-terminus make specific interactions with the substrate binding subsites. The active site of pepsinogen is intact; thus the lack of activity of pepsinogen is not due to a deformation of the active site. Nine ion pairs in pepsinogen may be important in the advent of activation and involve the activation peptide or regions of the mature N-terminus which are relocated in the mature enzyme. The activation peptide-pepsin junction, 44P-1, is characterized by high thermal parameters and weak density, indicating a flexible structure which would be accessible to cleavage. Pepsinogen is an appropriate model for the structures of other zymogens in the aspartic proteinase family.     

Activation of serum prophenoloxidase in arthropod immunity. The specificity of cell wall glucan activation and activation by purified fungal glycoproteins of crayfish phenoloxidase.

This study shows that the activation of crayfish serum prophenoloxidase by carbohydrates was specific for beta-1,3-glucans. Fractionation of the beta-1,3-glucan laminaran into laminaran M and laminaran G showed that both activated the proenzyme, but the G-chain had somewhat higher affinity for the proenzyme. Methylation analysis of these two fractions revealed that there were no 1,6-linkages present. Laminaripentaose, a linear pentasaccharide composed of (1 leads to 3)-linked beta-D-glucopyranosyl residues was also active but had a lower affinity for the proenzyme than laminaran G. Laminaran completely inhibited the activation of prophenoloxidase by the pentaose. In the concentrations tested, laminaran was not inhibitory to the phenoloxidase activity. Purified extracellular glycoproteins of the parasitic fungus Aphanomyces astaci also strongly activated crayfish serum prophenoloxidase. Only high molecular weight glycoproteins were effective. Exo-beta-1,3-glucanase treatment decreased the activating capacity, suggesting that at least part of the glycoproteins consisted of beta-1,3-glucans. The significance of these results in the defence against parasitic fungi in crayfish is discussed.     

Calcium-dependent activation of a multifunctional protein kinase by membrane phospholipids.

The proenzyme of a Ca2+-dependent protease-activated protein kinase previously obtained from mammalian tissues (Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616) was enzymatically fully active without limited proteolysis when Ca2+ and a membrane-associated factor were simultaneously present in the reaction mixture. The activation process was reversed by removing Ca2+ with ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid. An apparent Ka value for Ca2+ was less than 5 x 10(-5) M. Other divalent cations were inactive except for Sr2+, which was 5% as active as Ca2+. The factor was almost exclusively localized in membrane fractions of various tissues including brain, liver, kidney, skeletal muscle, blood cells, and adipose tissue. It was easily extractable with chloroform/methanol (2:1), and was recovered in the phospholipid fraction. In fact, this membrane factor could be replaced by chromatographically pure phosphatidylinositol, phosphatidylserine, phosphatidic acid, or diphosphatidylglycerol. Phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin were far less effective under the comparable conditions. Ca2+-dependent modulator protein was unable to support enzymatic activity. The enzyme thus activated showed an ability to phosphorylate five histone fractions and muscle phosphorylase kinase, and appeared to possess multifunctional catalytic activities.     

Identification, characterization and deduced amino acid sequence of the dominant protease from Kudoa paniformis and K. thyrsites: a unique cytoplasmic cysteine protease

Kudoa paniformis and Kudoa thyrsites (Myxozoa: Myxosporea) infections are associated with severe proteolysis of host muscle tissue post-mortem. The present study was undertaken to identify and characterize the protease responsible for myoliquefaction and determine mechanisms controlling protease function in vivo. N-terminal sequence analysis of partially purified protease from hake muscle infected with K. paniformis and K. thyrsites revealed a 23 amino acid sequence that aligned with cysteine proteases. Enzyme inhibition assays confirmed the presence of an essential active site cysteine residue. Using the above K. paniformis amino acid sequence data, a corresponding cDNA sequence from K. thyrsites plasmodia was elucidated revealing a cathepsin L proenzyme (Kth-CL). The translated amino acid sequence lacked a signal sequence characteristic of lysosomal and secreted proteins suggesting a unique cytoplasmic location. Only the proenzyme form of Kth-CL was present in Atlantic salmon muscle anti-mortem but this form became processed in vivo when infected muscle was stored at 4 degrees C. The proenzyme of Kth-CL showed uninhibited activity at pH 6.0, negligible activity at pH 6.5 and no measurable activity at pH 7.0 whilst the processed protease showed stability and function over a broad pH range (pH 4.5-8.8). The pH dependent processing and function of Kth-CL was consistent with histidine residues in the proregion playing a critical role in the regulation of Kth-CL.     

Mapping the pro-region of carboxypeptidase B by protein engineering. Cloning, overexpression, and mutagenesis of the porcine proenzyme.

The proteolytic processing of pancreatic procarboxypeptidase B to a mature and functional enzyme is much faster than that of procarboxypeptidase A1. This different behavior has been proposed to depend on specific conformational features at the region that connects the globular domain of the pro-segment to the enzyme and at the contacting surfaces on both moieties. A cDNA coding for porcine procarboxypeptidase B was cloned, sequenced, and expressed at high yield (250 mg/liter) in the methylotrophic yeast Pichia pastoris. To test the previous hypothesis, different mutants of the pro-segment at the putative tryptic targets in its connecting region and at some of the residues contacting the active enzyme were obtained. Moreover, the complete connecting region was replaced by the homologous sequence in procarboxypeptidase A1. The detailed study of the tryptic processing of the mutants shows that limited proteolysis of procarboxypeptidase B is a very specific process, as Arg-95 is the only residue accessible to tryptic attack in the proenzyme. A fast destabilization of the connecting region after the first tryptic cut allows subsequent proteolytic processing and the expression of carboxypeptidase B activity. Although all pancreatic procarboxypeptidases have a preformed active site, only the A forms show intrinsic activity. Mutational substitution of Asp-41 in the globular activation domain, located at the interface with the enzyme moiety, as well as removal of the adjacent 310 helix allow the appearance of residual activity in the mutated procarboxypeptidase B, indicating that the interaction of both structural elements with the enzyme moiety prevents the binding of substrates and promotes enzyme inhibition. In addition, the poor heterologous expression of such mutants indicates that the mutated region is important for the folding of the whole proenzyme.     

Dissociation of m-calpain subunits occurs after autolysis of the N-terminus of the catalytic subunit, and is not required for activation.

Calpain is a heterodimeric, intracellular Ca(2+)-dependent, "bio-modulator" that alters the properties of substrates through site-specific proteolysis. It has been proposed that calpains are activated by autolysis of the N-terminus of the large subunit and/or its dissociation into the subunits. It is, however, unclear whether the dissociation into subunits is required for the expression of protease activity and/or for in vivo function. Recently, the crystal structure of m-calpain in the absence of Ca(2+) has been resolved. The 3D structure clearly shows that the N-terminus of the m-calpain large subunit (mCL) makes contact with the 30K subunit, suggesting that autolysis of the N-terminus of mCL changes the interaction of both subunits. To examine the relationship between autolysis, dissociation, and activation, we made and analysed a series of N-terminal mutants of mCL that mimic the autolysed forms or have substituted amino acid residue(s) interacting with 30K. As a result, the mutant m-calpains, which are incapable of autolysis, did not dissociate into subunits, whereas those lacking the N-terminal 19 residues (Delta 19), but not those lacking only nine residues (Delta 9), dissociated into subunits even in the absence of Ca(2+). Moreover, both Delta 9 and Delta 19 mutants showed an equivalent reduced Ca(2+) requirement for protease activity. These results indicate that autolysis is necessary for the dissociation of the m-calpain subunits, and that the dissociation occurs after, but is not necessary for, activation.     

Activation of proenzyme of acidic protease from human seminal plasma.

The kinetics and the extent of the conversion of the proenzyme into the active acidic protease (EC 3.4.23.--) of human seminal plasma were dependent on acidic pH. Between pH 2 and 4, the initial rate of the activation was first-order with respect to the proenzyme. Between pH 4.5 and 5, the rate deviated from the first-order with an initial lag period which can be abolished by adding an excess amount of the acidic protease or pepsin. The extent of the activation was complete between pH 2 and 3 and became incomplete between pH 4 and 5. Addition of the acidic protease or pepsin did not alter the extent of the activation at the high pH values. According to the chromatographic profile on a Sephadex G-75 column, the activation products (namely active acidic protease and an activation peptide) obtained at pH 3 and those obtained at pH 4.5 were identical. The molecular weight of the activation peptide obtained at pH 3 was 6900; its amino acid composition was analyzed and compared with those of the proenzyme and the acidic protease. Remarkable similarity between the amino acid composition of the acidic protease and that of human pepsin was observed. In the presence of an excess amount of hemoglobin, the conversion of the proenzyme was self-activated and showed an initial lag period. Addition of acidic protease did not change the rate of self activation or the lag period.     

Differential scanning calorimetric study of carboxypeptidase B, procarboxypeptidase B and its globular activation domain

High-sensitivity differential scanning calorimetry has been applied to the study of porcine pancreatic carboxypeptidase B, the proenzyme and its 81-residue activation domain. The thermal study has been carried out over a range of scan rates, ionic strengths and pH values. The thermal unfolding of the isolated activation domain has been found to be reversible and corresponds to that of a typical compact globular structure, with melting temperatures higher than those of the enzyme and proenzyme. Both proteins, on the other hand, undergo an irreversible, highly scan-rate-dependent thermal denaturation under all the experimental conditions investigated. The denaturation of the enzyme at pH 7.5 and the proenzyme at pH 7.5 and 9.0 follows the two-state irreversible model [Sánchez-Ruiz, J.M., López-Lacomba, J.L., Cortijo, M. & Mateo, P.L. (1988) Biochemistry 27, 1648-1652]. Thus the kinetic constants and activation parameters of the denaturation process could be obtained and compared to those for other proteins, particularly those of the closely related carboxypeptidase A system.     

Calcium-activated neutral protease and its endogenous inhibitor Activation at the cell membrane and biological function

The structures of calcium-activated neutral protease (CANP) and its endogenous inhibitor elucidated recently have revealed novel features with respect to their structure-function relationship and enzyme activity regulation. The protease is regarded as a proenzyme which can be activated at the cell membrane in the presence of Ca²⁺ and phospholipid, and presumably regulates the functions of proteins, especially membrane-associated proteins, by limited proteolysis. Protein kinase C is hydrolysed and activated by CANP at the cell membrane to a cofactor-independent form. These results are reviewed and the possible involvement of CANP in signal transduction is discussed.     

Structural basis of proteolytic activation of L-phenylalanine oxidase from Pseudomonas sp. P-501

The mature form of l-phenylalanine oxidase (PAOpt) from Pseudomonas sp. P-501 was generated and activated by the proteolytic cleavage of a noncatalytic proenzyme (proPAO). The crystal structures of proPAO, PAOpt, and the PAOpt-o-amino benzoate (AB) complex were determined at 1.7, 1.25, and 1.35A resolutions, respectively. The structure of proPAO suggests that the prosequence peptide of proPAO occupies the funnel (pathway) of the substrate amino acid from the outside of the protein to the interior flavin ring, whereas the funnel is closed with the hydrophobic residues at its vestibule in both PAOpt and the PAOpt-AB complex. All three structures have an oxygen channel that is open to the surface of the protein from the flavin ring. These results suggest that structural changes were induced by proteolysis; that is, the proteolysis of proPAO removes the prosequence and closes the funnel to keep the active site hydrophobic but keeps the oxygen channel open. The possibility that an interaction of the hydrophobic side chain of substrate with the residues of the vestibule region may open the funnel as a putative amino acid channel is discussed.     

Histidine decarboxylase of Lactobacillus 30a. Hydroxylamine cleavage of the -seryl-seryl- bond at the activation site of prohistidine decarboxylase

Conversion of the pi subunit of prohistidine decarboxylase to the alpha beta subunits of the active enzyme proceeds by a nonhydrolytic, monovalent cation-dependent, serinolysis reaction in which the hydroxyl oxygen of serine 82 of the pi chain is incorporated into the carboxyl group at the COOH terminus (serine 81) of the beta chain. Serine-82 becomes the pyruvate residue at the NH2 terminus of the alpha chain (Recsei, P.A., Huynh, Q. K., and Snell, E.E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 973-977). The unusual reactivity of this particular -Ser-Ser- bond is demonstrated by its sensitivity to 1 M hydroxylamine, which cleaves the native proenzyme under mild conditions (pH 8.0, 37 degrees C) to yield a modified beta chain with serine hydroxamate at the COOH terminus (Ser-81) and a modified alpha chain containing serine (Ser-82 of the proenzyme) rather than pyruvate at the NH2 terminus. Neither an -Asn-Gly- bond nor other -Ser-Ser- bonds in the proenzyme were cleaved under these conditions. The reaction also did not occur with the denatured enzyme or with model peptides, indicating that the enhanced reactivity is a result of the particular conformation at this position in the native protein. The reaction with the native proenzyme proceeded optimally at pH 7.5-8.0 with a half-time (30 min) substantially less than that (3.5-4.5 h) required for the activation reaction and was not increased in rate by addition of K+. Correspondingly, preincubation of the proenzyme at pH 8.0 in the absence of both hydroxylamine and K+ modestly increased the rate of activation when K+ was subsequently added. Although these findings do not exclude other mechanisms, they are all consistent with and most easily explained by rearrangement of the pi chain to form an internal ester intermediate prior to the beta-elimination that occurs during activation to yield the alpha and beta chains of the mature enzyme.