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The gene encoding a subtilisin-like serine proteinase in the psychrotrophic Vibrio sp. PA44 has been successfully cloned, sequenced and expressed in Escherichia coli . The gene is 1593 basepairs and encodes a precursor protein of 530 amino acid residues with a calculated molecular mass of 55.7 kDa. The enzyme is isolated, however, as an active 40.6-kDa proteinase, without a 139 amino acid residue N-terminal prosequence. Under mild conditions the enzyme undergoes a further autocatalytic cleavage to give a 29.7-kDa proteinase that retains full enzymatic activity. The deduced amino acid sequence of the enzyme has high homology to proteinases of the proteinase K family of subtilisin-like proteinases. With respect to the enzyme characteristics compared in this study the properties of the wild-type and recombinant proteinases are the same. Sequence analysis revealed that especially with respect to the thermophilic homologues, aqualysin I from Thermus aquaticus and a proteinase from Thermus strain Rt41A, the cold-adapted Vibrio -proteinase has a higher content of polar/uncharged amino acids, as well as aspartate residues. The thermophilic enzymes had a higher content of arginines, and relatively higher number of hydrophobic amino acids and a higher aliphatic index. These factors may contribute to the adaptation of these proteinases to different temperature conditions. [ABSTRACT FROM AUTHOR]

It is scientifically and industrially important to clarify the stabilizing mechanism of proteases in extraordinary environments. We used subtilisins ALP I and Sendai as models to study the mechanism. Subtilisin ALP I is extremely sensitive to highly alkaline conditions, even though the enzyme is produced by alkalophilic Bacillus , whereas subtilisin Sendai from alkalophilic Bacillus is stable under conditions of high alkalinity. We constructed mutant subtilisin ALP I enzymes by mutating the amino acid residues specific for subtilisin ALP I to the residues at the corresponding positions of amino acid sequence alignment of alkaline subtilisin Sendai. We observed that the two mutations in the C-terminal region were most effective for improving stability against surfactants and heat as well as high alkalinity. We predicted that the mutated residues are located on the surface of the enzyme structures and, on thebasis of three-dimensional modelling, that they are involved in stabilizing the conformation of the C-terminal region. As proteolytic enzymes frequently become inactive due to autocatalysis, stability of these enzymes in an extraordinary environment would depend on the conformational stability of the molecular surface concealing scissile peptide bonds. It appeared that the stabilization of the molecular surface structure was effective to improve the stability of the proteolytic enzymes. [ABSTRACT FROM AUTHOR]

The disease Fusarium head blight (scab) causes severe problems for farmers and for the industries that use cereals. It is likely that the fungi that cause scab (Fusarium spp.) use various enzymes when they invade grains. We are studying enzymes that the fungi may use to hydrolyze grain proteins. To do this, Fusarium culmorum was grown in a gluten-containing medium from which an alkaline serine proteinase with a molecular mass of 28.7 kDa was purified by size-exclusion and cation exchange chromatographies. The enzyme was maximally active at pH 8.3–9.6 and 50 °C, but was unstable under these conditions. It hydrolyzed the synthetic substrates N -succinyl-Ala-Ala-Pro-Phe p -nitroanilide and, to a lesser extent, N -succinyl-Ala-Ala-Pro-Leu p -nitroanilide. It was inhibited by phenylmethanesulfonyl fluoride and chymostatin, but not by soybean trypsin or Bowman–Birk inhibitors. Parts of the amino-acid sequence were up to 82% homologous with those of several fungal subtilisins. One of the active site amino acids was detected and it occupied the same relative position as in the other subtilisins. Therefore, on the basis of these characteristics, the proteinase is subtilisin-like. Purification of the enzyme was complicated by the fact that, when purified, it apparently underwent autolysis. The presence of extraneous protein stabilized the activity. [ABSTRACT FROM AUTHOR]

Partial metal depletion and 113Cd-NMR studies have suggested that the recombinant Cd-containing metallothionein of the sea urchin Strongylocentrotus purpuratus (Cd7-MTA) binds its metal ions in a four-metal (Cd4Cys,11) and a three-metal (Cd7Cys9) cluster associated with the N-terminal and C-terminal halves of the protein, respectively [Wang, Y., Mackay, E. A., Zerbe, O., Hess, D., Hunziker, R E., Vasak, M. & Kägi. J. H. R. (1995) Biochemistry 34, 7460–7467]. This partitioning has now been confirmed by bisecting native Cd?-MTA with subtilisin into products bearing only a single metal-thiolate cluster. Their separation by reverse-phase HPLC and on-line electrospray mass spectrometry in combination with sequence analysis revealed selective cleavage of the protein into a set of N-terminal polypeptides containing 37–39 residues with four Cd ions and a set of C terminal polypeptides containing 24 and 25 residues with three Cd ions. Thus, sea urchin MTA like its mammalian counterparts is made up of two separate cluster-harboring domains. The fragmentation pattern indicated that the sites of cleavage are located in the peptide loop interspaced between the first two metal bound cysteine residues of the C-terminal domain. Accordingly, with cleavage, one of the putative nine thiolate ligands of the three-metal cluster was lost to the N terminal fragment. The coordinational consequences of this repartition were reflected in massive chiroptical changes accompanying the cleavage process. While the liberated N-terminal domain retained the CD profile of the four-metal cluster in the parent protein and thereby indicated preservation of its structure, the CD features attributable to the intact three-metal cluster were largely lost on cleavage. The vanished features bear strong resemblance to the large biphasic ellipticity signal at 250 nm which dominates the CD spectrum of native Cd7 MTA, and allow us thus to attribute this signal to excitonic coupling interactions of Cd-thiolate chromophores in the three-metal cluster. [ABSTRACT FROM AUTHOR]

Backbone dynamics of Savinase, a subtilisin of 269 residues secreted by Bacillus lentus, have been studied using 15N relaxation measurements derived from proton-detected two-dimensional 1H-15N-NMR spectroscopy. 15N spin-lattice rate constants (R1), spin-spin relaxation-rate constants (R2), and 1H-15N nuclear Overhauser effects (NOE) were determined for 84% of the backbone amide 15N nuclei. The model-free formalism [Lipari, G. & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546–4559] was used to derive values for a generalized order parameter, S2, interpretable as a measure of the amplitude of motion on the picosecond-nanosecond timescale, for each N-H bond vector. Additional terms used to fit the data include an effective correlation time for internal motions (τe) and an exchange term (Rex) to account for exchange contributions to R2. The overall rotational correlation time (τm) is 9.59 ± 0.02 ns; the average order parameter (S2) is 0.90 ± 0.07, indicative of a rigid structure consistent with Savinase's high degree of secondary structure and compact tertiary fold. Residues S125-S128, located in the substrate-binding region, represent the longest stretch of protein which exhibits disorder on the picosecond-nanosecond timescale. These residues also exhibit significant exchange terms, possibly indicative of motion on the microsecond-millisecond timescale, which could also be influenced by the proximity of the phenyl ring of the substituted aryl boronic acid inhibitor used in this study. S103 and G219 in the substrate-binding region also show flexibility on the picosecond-nanosecond timescale. There is also significant motion in the turn, G258-T260, of a small solvent-exposed loop region which may make the protein vulnerable to autolysis at that point. Some residues in both calcium-binding sites and nearby also show mobility. [ABSTRACT FROM AUTHOR]

The effect of high pH (pH 10.5) on Savinase, a subtilisin-like serine proteinase, has been investigated using X-ray crystallography. The structures of two Savinase mutants were determined at two different pH values, namely pH 6.0, where the enzyme is inactive (this is the pH at which most of the structural work has been carded out on other serine-proteinases), and pH 10.5. where Savinase is active. Comparison of these high resolution (0.16 nm) structures showed four related sets of changes between the two pH values. First, the difference in protonation state of the active-site histidine leads to a change in conformation of the active-site triad. Secondly, there are resulting changes in the water structure around this histidine residue with much increased mobility of the water in the active-site at pH 10.5. Thirdly, the two substrate-binding loops on either side of the by ordered water molecules at pH 10.5 and show substantially binding site are less well linked increased flexibility. Finally, the first substrate-binding loop, residues at positions 99-104, moves slightly so as to widen the substrate channel. [ABSTRACT FROM AUTHOR]

A model is presented for the three-dimensional structure of the catalytic domain of the human serine proteinase furin and its interaction with model substrates. This homology model is based on the crystal structures of subtilisin BPN' and thermitase in complex with the inhibitor eglin, and it also applies to other members of the eukaryotic subtilisin-like proprotein convertases. Predictions are made of the general protein fold, inserted loops, disulfide bonds, Ca2+-binding sites and salt bridges. A detailed prediction of the substrate-binding region attempts to explain the basis of specificity for multiple basic residues preceding the cleavage site. Specific acidic residues in the S, S2 and S4 subsites of the substrate-binding region of furin are identified which appear to be of particular importance, while residues of the S2′, S3, S5 and S6 subsites may also contribute to substrate binding. Based on this method, protein engineering can be employed not only to test the predicted enzyme-susbtrate interactions, as demonstrated for human furin, but, equally importantly, to design proprotein convertases with a desired specificity, or to design novel substrates or inhibitors. [ABSTRACT FROM AUTHOR]

The major coat protein of native filamentous phage fd is vulnerable to digestion by subtilisin, but not by any of a number of other proteolytic enzymes. Degradation by the non-specific protease subtilisin occurs at specific sites in the N-terminal portion of g8p. The N-terminal part of the protein is considered to be the outer layer of a two-layered coat. Thus, subtilisin treatment results in a monolayered phage particle. These particles possess the morphology and stability of native phage fd. Furthermore, subtilisin proteolysis proved to be an efficient instrument in detecting variations in the topology of the g8p of related filamentous phages. [ABSTRACT FROM AUTHOR]

The previous notion that the amino acid side chain at position 104 of subtilisins is involved in the binding of the side chain at position P4 of the substrate has been investigated. The amino acid residue Val104 in subtilisin 309 has been replaced by Ala, Arg, Asp, Phe, Ser, Trp and Tyr by site-directed mutagenesis. It is shown that the P4 specificity of this enzyme is not determined solely by the amino acid residue occupying position 104, as the enzyme exhibits a marked preference for aromatic groups in P4, regardless of the nature of the position-104 residue. With hydrophilic amino acid residues at this position, no involvement is seen in binding of either hydrophobic or hydrophilic amino acid residues at position P4 of the substrates. The substrate with Asp in P4 is an exception, as the preference for this substrate is increased dramatically by introduction of an arginine residue at position 104 in the enzyme, presumably due to a substrate-induced conformational change. However, when position 104 is occupied by hydrophobic residues, it is highly involved in binding of hydrophobic amino acid residues, either by increasing the hydrophobicity of S4 or by determining the size of the pocket. The results suggest that the amino acid residue at position 104 is mobile such that it is positioned in the S4 binding site only when it can interact favourably with the substrate's side chain at position P4. [ABSTRACT FROM AUTHOR]

Lactococcus lactis strains produce an extracellular subtilisin-related serine proteinase in which immunologically different components can be distinguished. Monoclonal antibodies specific for the different proteinase components have been raised and their epitopes were identified. By Western-blot analysis it was found that all monoclonal antibodies recognize ail denatured proteinase components. The distinction between the different components could be made under native conditions only, indicating that binding regions are masked in the native molecule. In a L. lactis proteinase which was inactivated by the substitution Asp30 → Asn under native conditions, only one epitope could be detected. This demonstrates that autoproteolytic activity is required to make specific binding regions accessible for (monoclonal) antibodies. [ABSTRACT FROM AUTHOR]

A juvenile-hormone-binding protein (juvenile-hormone carrier), isolated from Galleria meionella haemolymph, was treated with trypsin, chymotrypsin, carboxypeptidase A and subtilisin. Among these enzymes, only subtilisin was able to affect juvenile-hormone-binding activity of this protein. With SDS/PAGE it was shown that juvenile- hormone-binding protein, a 32-kDa peptide, is first slowly converted into a 30-kDa molecule, then into two or three smaller-molecular-mass species (20-25 kDa), which in turn were further digested to small peptides undetectable in PAGE. The 30-kDa peptide has a 2.4-times-higher dissociation constant for juvenile hormone than the native protein. No binding activity was detected for 20- 25-kDa peptides. The rate of proteolysis of juvenile-hormone- binding protein was decreased by more than twofold in the presence of hormone, however, the overall cleavage pattern was unchanged. Under non-denaturing conditions, free binding-protein molecules could be separated from juvenile-hormone - binding-protein complex due to a slower electrophoretic mobility of the complex. As judged from ultracentrifugation and cross-linking experiments, binding of the hormone to its baemolymph carrier does not induce formation of oligomers, but shifts the sedimentation coefficient from 2.30S to 2.71 S. It is concluded that juvenile-hormone binding induces a conformational transition of its carrier protein. This hormone-induced change might have a physiological significance for signal transmission. [ABSTRACT FROM AUTHOR]

The subtilisins are known to be susceptible to chemical oxidation due to the conversion of Met222 into the corresponding sutfoxide. A number of derivatives with resistance towards oxidation have previously been prepared by replacement of this group with the other 19 amino acid residues. Unfortunately, the activities of these enzymes were of the order of l - 10% of that obtained with the wild-type enzyme, in contrast, the oxidation-labile cysteine mutant exhibited much higher activity, suggesting that this is associated with the presence of a sulphur atom in the amino acid at position 222. It is shown here that it is possible to maintain a sulphur atom in the amino acid at position 222 without the enzyme becoming labile towards oxidation. A subtilisin from Bacillus lentus, subtilisin 309, in which Met222 was replaced with a cysteinyl residue by site-directed mutagenesis was modified with thioalkylating reagents. Treatment of such enzyme derivatives with H2O2 revealed that their stabilities towards oxidation had increased significantly compared to both wild-type and unmodified [Cys222]subtilisin. One of the chemically modified enzyme derivatives, [Me-S-Cys222]subtilisin, exhibited a kcat/Km value of 56% of that obtained with the wild-type enzyme when assayed against the substrate Suc-Ala-Ala-Pro-Phe-NH-Ph-NO2 (Suc, succinyl) and it exhibited 89% activity when tested in an assay with dimethyl casein as a substrate. The corresponding values obtained for unmodified [Cys222subtilisin were lower, i.e. 39% for the dimethyl casein activity and 46% for the kcat/Km for the hydrolysis of Suc-Ala-AlaPro-Phe-NH-Ph-NO2. This demonstrates the feasibility of 'replacing the oxidation-labile methionyl residue group in a subtilisin enzyme with a group stable towards oxidation without substantially reducing the activity. [ABSTRACT FROM AUTHOR]

Triclinic crystals of the complex formed by eglin with subtilisin Carlsberg were analyzed by X-ray diffraction. The crystal and molecular structure of this complex was determined with data that extended to 0.12-rim resolution by a combination of Patterson search methods and isomorphous replacement techniques. Its structure was refined to a crystallographic R value of 0.178 (1.0-0.12 nm) using an energy-restraint least-squares procedure. The complete subtilisin molecule could be traced without ambiguity in the refined electron density. The eglin component, from which an amino-terminal segment is cleaved off, is only defined from Lys81 (i.e. the lysine residue 8 of the inhibitor) onwards. Per unit cell, 436 fixed solvent molecules and 2 calcium ions were located. In spite of 84 amino acid replacements and one deletion, subtilisin Carlsberg exhibits a very similar polypeptide fold to subtilisin BPN'. The root-mean-square deviations of all &alpha-carbon atoms (excluding those at the deletion site) from models of subtilisin BPN' [Alden, R. A., Birktoft, J. J., Kraut, J., Robertus, J. D. & Wright, C. S. (1971) Biochem. Biophys. Res. Commun. 45, 337-344] and subtilisin Novo [Drenth, J., Hol, W. G. J., Jansonius, J. N. & Kockoek, R. (1972) Eur. J. Biochem. 25, 177-181] are 0.077 nm and 0.103 nm. Most of these deviations result from global shifts rather than changes of the local geometry. The single-residue deletion at position 56 affects only "the surrounding conformation. Two sites of high electron density and close distances to surrounding oxygen ligands have been found in the Carlsberg enzyme which are probably occupied by calcium ions. Eglin consists of a twisted four-stranded β-sheet flanked by an α-helix and by an exposed proteinase binding loop on opposite sides. Around the reactive site, Leu45I-Asp46I, this loop is mainly stabilized by electrostatic/ hydrogen bond interactions with the side chains... [ABSTRACT FROM AUTHOR]

Limited subtilisin cleavage of tubulin results in formation of S-tubulin heterodimer and a 4-kDa carboxyl-terminal peptide fragment. This carboxyl-terminal domain constitutes an essential site for MAPs interaction and plays a role in modulating the interactions responsible for tubulin self-assembly into microtubules [Serrano et al. (1984) Proc. Natl Acad. Sci. USA 81, 5989; and Biochemistry 23, 4675]. In the present communication it is shown that addition of the 4-kDa peptide fragment from porcine tubulin to porcine S-tubulin in a molar ratio of about 2:1 does not affect the assembly of the latter. On the other hand, consistent with previous findings on the binding of the 4-kDa peptide by MAP-2, the peptide inhibited MAP-2-induced tubulin assembly (molar ratio of peptide to tubulin, about 2:1; peptide to MAP-2, about 30:1). Comparison of the amino acid composition of the 4-kDa peptide fragment and the C-terminal amino acid residues of S-tubulin with the amino acid sequence of tubulin indicated the subtilisin cleavage site on the tubulin molecule to be between residues Glu417 and Phe418 of the α-subunit sequence and between Glu407 and Phe408 of the β-subunit sequence. The circular dichroism of the 4-kDa fragment in water as solvent is indicative of a molecule with an unordered structure, but when the solvent is changed to a water-trifluoroethanol mixture, the fragment becomes more highly structured. The critical concentration for S-tubulin assembly is not affected by MAPs nor by polylysine, but is decreased by either taxol of dimethylsulfoxide. S-tubulin, with its greater propensity for self-association, has a different conformation from tubulin as shown by a 50% decrease in α-helical content, a more hydrophobic environment of at least some of the tryptophan residues as judged from fluorimetry, and a greater compaction indicated by f/f0 = 1.3, as compared to 1.4 for tubulin. The latter point is supported by the observation that the value of the sedimentation coefficient, s20,w = 5.7 S, of the 92-kDa S-tubulin molecule is not significantly different from that of the 100-kDa tubulin, s20,w = 5.8 S. [ABSTRACT FROM AUTHOR]

Limited proteolysis of tubulin with subtilisin resulted in the removal of the carboxyl-terminal moiety of tubulin subunits. The remaining peptides from both α and β tubulin lacking the carboxyl terminal did not bind to tau factor nor to MAP2 or MAP1. The carboxyl-terminal fragments bind to tau factor and MAP2 and both compete for the same binding sites in the tubulin molecule. Our results suggest that the carboxyl-terminal region of tubulin is a regulatory domain for the assembly of tubulin and the site for interaction with MAPs. [ABSTRACT FROM AUTHOR]

The crystal structure of subtilisin DY inhibited by N-benzyloxycarbonyl-Ala-Pro-Phe-chloromethyl ketone has been solved by molecular replacement with subtilisin Carlsberg as the starting model. The model has been refined to a crystallographic R factor (= Σ ∣ ∣Fo∣ - ∣Fc∣ ∣ / Σ ∣Fo∣) of 15.1 % using X-ray diffraction data to 0.175 nm resolution. Subtilisin DY is an alkaline proteinase from the X-irradiated Japanese strain DY of Bacillus licheniformis, which normally produces subtilisin Carlsberg. It has very similar properties to subtilisin Carlsberg, with a slightly enhanced resistance to heat and guanidine hydrochloride-induced denaturation, in spite of the fact that the sequences of the two enzymes differ in 31 positions out of 274 residues. The close similarity in overall three-dimensional structure of subtilisins DY and Carlsberg and also their physicochemical properties, such as activity and stability, shows that nature aided by X-irradiation for rapid ’evolution' is able to accommodate considerable changes in sequence without substantial changes in property. [ABSTRACT FROM AUTHOR]

The course of inactivation of the reduced spinach chloroplast fructose-1,6-bisphosphatase by digestion with subtilisin has been followed by the progress curve method [Tsou, C. L. (1988) Adv. Enzymol. 61, 381-436] and found to follow first-order kinetics. On the basis of the hydrolysis of the substrate, fructose 1,6-bisphosphate, at different concentrations during proteolysis by subtilisin, the first-order inactivation rate constants for the free enzyme and the enzyme-substrate complex can both be determined. The ratio between the inactivation rate constants for the free enzyme and the enzyme-substrate complex indicates strong protection against subtilisin proteolysis by the substrate. It is proposed that the above ratio can be used as a quantitative measure of substrate protection for enzyme inactivation generally. As it has been found that the site of proteolysis is located in a loop region near the N-terminus and well away from the active site, the substrate protection indicates a conformation change of the enzyme away from the substrate binding site. [ABSTRACT FROM AUTHOR]

The chemical modification of bovine pancreatic ribonuclease A by 6-chloropurine 9-β-D-ribofuranosyl 5′-monophosphate was studied under several reaction conditions. The reaction, at pH 7.3, 40°C and a nucleotide:enzyme molar ratio of 60, showed a high degree of specificity in comparison to those corresponding to the base or the nucleoside. The main derivative was isolated by means of CM-cellulose chromatography. Subtilisin cleavage of this derivative showed that the substitution had taken place in the S-peptide moiety. Tryptic digestion of the S-peptide indicated that a lysine residue had been modified. Enzymatic and physico-chemical considerations showed that the actual site of reaction was the α-amino group of Lys-1. The structural and kinetic properties of the derivative are consistent with the existence of a phosphate-binding sub-site near the N-terminal region of the enzyme. [ABSTRACT FROM AUTHOR]

1. The kinetic parameters of 25 peptidyl-p-nitroanilide substrates were investigated with subtilisin Carlsberg as model enzyme. 2. For a series of 12 substrates, the contribution of various side chains to the affinity constant was computed by regression analysis. From these contributions the sequence of a new and better substrate, N-benzyloxycarbonyl-arginyl-norleucyl-norleucyl-p-nitroanilide (Z-Arg-Nle-Nle-Nan) was predicted. The compound was synthesized and assayed. Its calculated 1/Km value, 43.5 mM-1, was in a good agreement with the value of 40.0 mM-1 that was determined experimentally. 3. On expanding the series to 19 substrates, it was found that the productivity of enzyme-substrate binding is influenced primarily by those subsites which have a significantly greater contribution to the affinity constants than others. 4. The additivity principle applied reasonably well for the contribution of individual side chains to the kinetic parameters. This fact suggests that regression analysis can be used for the prediction of the amino acid sequence of better substrates than those already tested, probably not only for subtilisin but also for other proteolytic enzymes. [ABSTRACT FROM AUTHOR]

The time course of digestion of porcine thyroglobulin with subtilopeptidase at an enzyme to substrate ratio of 1 :200 was analyzed by sodium dodecylsulfate/polyacrylamide gel electrophoresis after reduction with dithiothreitol. Two fragments of Mr 155000 and 135000 were formed which showed a precursor-product relationship, the 135000-Mr species accumulating between 1 and 4h of digestion. After purification by elution from electrophoretic gels, these fragments were treated by CNBr and analyzed by dodecylsulfate/poiyacrylamide gel electrophoresis. Both mixtures of CNBr peptides showed a closely similar electrophoretic band pattern. These results suggest the presence in the thyroglobulin chain (Mr 330000) of half-sized segments each formed of a domain with strong tertiary structure of Mr about 140000 associated through the continuity of the chain to a less structured fragment of Mr about 20000. A 15000-Mr peptide was purified by Sephadex G-200 gel filtration from CNBr-treated thyroglobulin. Its uniqueness was demonstrated by N-terminal sequence analysis and by tryptic fingerprinting. Using densitometer recording of stained electrophoretic gels in sodium dodecylsulfate, the molar ratio of this peptide was estimated to be 2 mol/mol of 12-S thyroglobulin chain. These results are consistent with a protein structure in which two domains of similar size and structure are linked to form a single chain. Thus, the possibility exists that the structural gene for thyroglobulin might have evolved from duplication and fusion of an half-sized ancestral gene. [ABSTRACT FROM AUTHOR]

The reactive mercaptide-imidazolium ion-pair at the active site of papain and thiolsubtilisin was alkylated with negatively charged reactants. The reactivities of the two thiolenzymes differ considerably. The iodoacetate reaction is faster by more than 1000 times with papain than with thiolsubtilisin. On the other hand, towards 3-iodopropionate thiolsubtilisin is more reactive than papain by about a factor of 7. These findings, which are interpreted in terms of the different geometries of the two ion-pairs, offer an explanation of the basic difference between the catalytic abilities of papain and thiolsubtilisin. [ABSTRACT FROM AUTHOR]

Specific and non-specific trypsin substrates of known structure have been examined for common features. This analysis suggests that trypsin has a specificity for a particular conformation near the scissile bond which I denote as a conformational specificity. This conformation is a bent left-handed helix at the third and fourth (P3 and P4) amino acid positions toward the amino terminus from the scissile bond which I denote as a conformational specificity. This conformation is a bent left-handed show a high frequency of proline and glycine at these positions consistent with the left-handed helical conformation. This apparent secondary specificity for a particular substrate residue other than that at the primary position is not related to the nature of the residues at the third and fourth positions. Rather, these residues determine the bend of left-handed helix which has the effect of exposing main chain hydrogen-bonding groups of the substrate peptide chain to hydrogen-bonding groups on the enzyme. Thus, the secondary specificity of trypsin is not sequence-specific, but is for peptide main chain in the third and fourth positions and is determined by the tertiary structure of the substrate. This hypothesis for conformational and secondary specificity in trypsin can be extended to chymo- trypsin. It also provides a means for the regulation of certain processes in vivo catalyzed by other proteases. [ABSTRACT FROM AUTHOR]

It is known that each subunit of the tetrameric flavocytochrome b2 can be cleaved by yeast proteases to fragments of molecular weight 33–36000 and 21000, with some modification of catalytic properties, but without destruction of the oligorneric state of the protein. We report here experimental conditions which enabled us to simulate this specific cleavage in a controlled fashion with chymotrypsin and subtilisin. With trypsin and papain, on the other hand, it was not found possible to stop the digestion in such a way as to obtain a homogeneous still active product. A characterization of the enzymatic forms obtained by digestion with chymotrypsin and subtilisin at 0 °C shows that modification of enzymatic and solubility properties occurs in a stepwise fashion. It is also concluded that cleavage by yeast proteases is accompanied by loss of 10 to 25 residues. At 37 °C, chymotrypsin digestion yields a herne-binding core of molecular weight 15000, larger than the already characterized tryptic heme-binding core by about 40 residues. Although the latter is known to be very similar to trypsin-solubilized cytochrome b5, the lack of aggregation of the former in aqueous solution, its amino acid composition and circular dichroism spectra do not point to a similarity of its additional peptide segment with the hydrophobic tail of detergent-solubilized cytochrome b5. [ABSTRACT FROM AUTHOR]

Treatment of purified tryptophanyl-tRNA synthetase with either chymotrypsin, papain, subtilisin or elastase converts all the enzyme into a high-molecular-weight intermediate. This protease- resistant core molecule has the same dimeric structure as the native protein and possesses the ability to bind substrates (tryptophan, ATP and tRNATrp) but is catalytically inactive. The monomer molecular weight of the protease-treated enzyme is 39000 compared to 54000 for the intact molecule. Chemical studies indicate that proteases excise the amino-terminal part of the polypeptide chain. It has been demonstrated previously that removal of a 13000-dalton fragment from the amino-terminal region of the tryptophanyl-tRNA synthetase converts the native enzyme to another active form. Cleavage of 20 additional amino acids produces the inactive protease- resistant core. [ABSTRACT FROM AUTHOR]

Yeast phosphofructokinase having a molecular weight of 750000-800000 (20 S) has been subjected to limited proteolysis by subtilisin and yeast proteases. Two steps of proteolytic degradation could be distinguished: in the first step, which is accompanied by an increase in molecular activity, the subunits α and β (Mr 120 000) are converted to α' and β' (Mr approximately 90 000), and in the second step, accompanied by a decrease in enzyme activity, α' is converted to α' (Mr 80 000) and two further fragments having Mr 45 000 and 35 000 become detectable. In the course of this conversion the sedimentation value of the undissociated enzyme drops from 20 S to about 17 S. The two substrates fructose 6-phosphate and ATP exhibit characteristic protective effects on enzyme activity and on subunit degradation. Whereas the first step is not strongly influenced by the substrates, fructose, 6-phosphate inhibits significantly the degradation of α' and β', whereas ATP prevents only degradation of β'. When in presence of ATP α' is degraded to α'', the quaternary structure of the 17-S enzyme is no longer stable and a dissociation of this molecule occurs to a 12-S form which is enzymically active and ATP-sensitive and in which the ratio of α'' to β' is one-to-one. [ABSTRACT FROM AUTHOR]

Some analogues have been prepared of S-peptide, the peptide obtained together with S-protein from subtilisin-modified beef pancreatic RNase A. The syntheses are described of [Orn10, Asn14]- S-peptide and 1∊,7δ-triguanidino-[Orn10, Asn14]-S-peptide. The S-peptide analogues are able to activate S-protein at the level of the parent [Orn10]-S-peptide and 1∊ ,7∊-diguanidino-S-peptide respectively, although at high peptide-to-protein molar ratios. After their recombination with S-protein the buried character of Tyr-25 was restored, as judged from difference absorption and circular dichroism spectra in the near-ultraviolet region. These findings indicate that the asparaginyl residue is a possible naturally occurring substituent in the RNase A sequences whose state of amidation in position 14 has not yet been defined. [ABSTRACT FROM AUTHOR]

Studies the nitrogen-terminal amino-acid sequences of DNA polymerase I from Escherichia coli and of the large and the small fragments obtained by a limited proteolysis. Amino-acid composition of the native enzyme; Purification of the DNA polymerase I; Cleavage of native DNA polymerase I by subtilisin.

It is known that each subunit of the tetrameric flavocytochrome b2 can be cleaved by yeast proteases to fragments of molecular weight 33–36000 and 21000, with some modification of catalytic properties, but without destruction of the oligorneric state of the protein. We report here experimental conditions which enabled us to simulate this specific cleavage in a controlled fashion with chymotrypsin and subtilisin. With trypsin and papain, on the other hand, it was not found possible to stop the digestion in such a way as to obtain a homogeneous still active product. A characterization of the enzymatic forms obtained by digestion with chymotrypsin and subtilisin at 0 °C shows that modification of enzymatic and solubility properties occurs in a stepwise fashion. It is also concluded that cleavage by yeast proteases is accompanied by loss of 10 to 25 residues. At 37 °C, chymotrypsin digestion yields a herne-binding core of molecular weight 15000, larger than the already characterized tryptic heme-binding core by about 40 residues. Although the latter is known to be very similar to trypsin-solubilized cytochrome b5, the lack of aggregation of the former in aqueous solution, its amino acid composition and circular dichroism spectra do not point to a similarity of its additional peptide segment with the hydrophobic tail of detergent-solubilized cytochrome b5. [ABSTRACT FROM AUTHOR]

1. Maleylation of ε-amino groups of elastase and subtilisin causes a complete loss of their elastolytic activities while their esterolytic activities are almost unaffected. This results from a change in their electrostatic properties and is expressed in a complete loss of their ability to be adsorbed on elastin. The proteolytic activity of maleylated subtilisin toward casein and hemoglobin and V toward protamine sulphate did not change, but the Km value for the last substrate was significantly increased. The proteolytic activity of maleylated elastase toward casein and hemoglobin was reduced respectively to 30 and 70%, while the activity toward protamine sulphate was increased as a result of a ten-fold decrease m Km value. These results indicate that ε-amino groups of elastase serve as secondary binding sites. 2. Elastolytic activity of 2,3-dimethyl-maleylated elastase and subtilisin could be recovered by hydrolysis of the 2,3-dimethyl-maleylamino bonds, at room temperature, pH 6.9. 3. Acetyl-L-alanyl-L-alanyl-L-alanine methyl ester was found to be an excellent esteratic substrate for subtilisin. The apparent Km and kcat values at 30 °C, pH 8.0 are 1.7 mM, and 1190 sec-1, respectively. [ABSTRACT FROM AUTHOR]

Treatment of native DNA polymerase from Escherichia coli with subtilisin may lead to its cleavage into a polymerase fragment, and an exonuclease fragment. The cleavage has been followed by electrophoresis in polyacrylamide gel in the presence of sodium dodecylsulfate and by the change both in polymerase activity and in exonuclease activity. The electrophoretic analysis has shown that subtilisin treatment leads to the formation of two new protein bands and simultaneous disappearance of the band corresponding to the native enzyme. The cleavage of the native polymerase may be accompanied by change in the polymerase activity. The change appears to depend on the conditions of the polymerase assay. With activated calf thymus DNA the activity increases by a factor of up to 2.3 when assayed at low phosphate buffer concentration. At higher buffer concentration in the assay mixture the cleavage gives rise to a decrease in polymerase activity. With poly[d(A-T)] · poly[d(A-T)] the cleavage gives rise to a decrease in polymerase activity at both low and high buffer concentration. The effect of buffer concentration on the polymerase activity of the native and the cleaved enzyme showed that maximum activity for the two enzymes was obtained at different buffer concentrations in the presence of activated calf thymus DNA. In the presence of poly[d(A-T)] · poly[d(A-T)] maximum activity was obtained at the same buffer concentration but the activity of the cleaved enzyme was lower than that of the native at all buffer concentrations. A similar differential effect of ionic strength on the activity has been found for the exonuclease site of the native enzyme and the cleaved exonuclease fragment. This differential effect depends, however, on the molecular size of the substrate poly[d(A-T]) · poly[d(A-T)]. It appears, furthermore, that the exonuclease site of the cleaved exonuclease fragment has a smaller affinity towards poly[d(A-T)] · poly[d(A-T)] than that of the native enzyme. Finally, while the exonuclease activity of the native enzyme towards poly[d(A-T)] · poly[d(A-T)] is stimulated by conditions that permit simultaneous synthesis no such synthesis stimulated exonuclease activity is observed after cleavage of the native enzyme into a polymerase fragment and an exonuclease fragment. [ABSTRACT FROM AUTHOR]

The reaction of thiolsubtilisin with iodoacetamide shows that in the alkaline pH-range the —SH group of the enzyme reacts like a simple thiol compound such as mercaptoacetate. On the other hand, around neutral pH the enzyme displays enhanced reactivity compared with that of mercaptoacetate. On the basis of measurements in ²H2O it is concluded that a mercaptideimidazolium ion-pair is formed in thiolsubtilisin and this accounts for the enhanced reactivity around neutral pH. Iodoacetamide reacts with the —SH group of the enzyme, as compared to mercaptoacetate, at a relatively higher rate than bromo-, chloro-, and fluoroacetamide. This might be due to the less polar environment around the —SH group on the surface of the enzyme than in water, as indicated by alkylation of mercaptoacetate with iodo- and chloroacetamide in the presence of dioxane. By alkylating thiolsubtilisin with enantiomeric reactants, a slight conformational change at the active site, not measurable with ordinary physical methods, could be establisbed above pH &assymp; 9, whereas the overall protein structure is stable up to pH &assymp; 11. D-2-Bromo-n-butyramide reacts with thiolsubtilisin about 60 times as fast as D-2-bromopropionamide. This indicates that hydrophobic interaction between the enzyme and a methyl group of the substrate can enhance the reaction rate by more than one order of magnitude. Model building of the active site suggests that Ala-152 and Thr-220 of thiolsubtilisin may form van der Waals interactions with D-2-bromo-n-butyramide if the alkylating agent is properly positioned for reaction. No such interaction is possible between D-2-bromopropionamide and thiolsubtilisin. [ABSTRACT FROM AUTHOR]

The three-dimensional structure of subtilisin Novo was determined by X-ray crystallographic methods to a resolution of 0.28 nm and compared with the structure of subtilisin BPN′ The two subtilisins have an equal amino acid sequence. It is shown that neither the quite different media nor the different lattice contacts in the two crystal forms result in any major structural change. Subtilisin Novo has a binding site for monovalent cations. [ABSTRACT FROM AUTHOR]