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At least 19 recordsLinked to original sources
PubMed1978

The determination of molecular weights of Streptomyces subtilisin inhibitor and the complex of Streptomyces subtilisin inhibitor and subtilisin BPN' by sedimentation equilibrium.

The molecular weight of Streptomyces subtilisin inhibitor (SSI), a protein proteinase inhibitor, and that of the complex of SSI and subtilisin BPN' [EC 3.4.21.14] were determined by a sedimentation equilibrium method in 25 mM phosphate buffer, at pH 7.0, ionic strength 0.1 M (NaCl), 25.0 degrees C. The molecular weight of SSI was found to be 23,000 over a wide concentration range, 0.01-10 mg/ml, the range used for inhibitory, spectrophotometric, and kinetic measurements. Based on the amino acid sequence, the molecular weight of SSI has been calculated to be 11,500 (Ikenaka, T., et al. (1974) J. Biochem. 76, 1191-1209); therefore, the molecular weight of 23,000 obtained above suggests that SSI is in a dimeric form under usual conditions in the concentration range of 5 X 10(-7)-5 X 10(-4) M. The molecular weight of the subtilisin BPN'-SSI complex was determined to be 78,000 in the concentration range of 0.03-5.0 mg/ml by sedimentation equilibrium of the crystallized preparation and by that of a mixture of subtilisin BPN' and SSI treated as a multicomponent-polydisperse system. The molecular weight obtained here, combined with the results of binding stoichiometry (Inouye, K., et al. (1977) J. Biochem. 82, 961-967) that showed that one mol of SSI (molecular weight, 11,500) and one mol of the enzyme (molecular weight, 27,500) are tightly bound (Kd less than 1 nM), demonstrate that one mol of dimeric SSI binds two mol of the enzyme to form a stable complex, E2I2.

Bacterial Proteins↗
PubMed1991

Refined crystal structure of the complex of subtilisin BPN' and Streptomyces subtilisin inhibitor at 1.8 A resolution.

The crystal structure of subtilisin BPN' complexed with a proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor) was refined at 1.8 A resolution to an R-factor of 0.177 with a root-mean-square deviation from ideal bond lengths of 0.014 A. The work finally established that the SSI-subtilisin complex is a Michaelis complex with a distance between the O gamma of active Ser221 and the carbonyl carbon of the scissile peptide bond being an intermediate value between a covalent bond and a van der Waals' contact, 2.7 A. This feature, as well as the geometry of the catalytic triad and the oxyanion hole, is coincident with that found in other highly refined crystal structures of the complex of subtilisin Novo, subtilisin Carlsberg, bovine trypsin or Streptomyces griseus protease B with their proteinaceous inhibitors. The enzyme-inhibitor beta-sheet interaction is composed of two separate parts: that between the P1-P3 residues of SSI and the 125-127 chain segment (the "S1-3 site") of subtilisin and that between the P4-P6 residues of SSI and th 102-104 chain segment (the "S4-6 site") of subtilisin. The latter beta-interaction is unique to subtilisin. In contrast, the beta-sheet interaction previously found in the complex of subtilisin Novo and chymotrypsin inhibitor 2 or in the complex of subtilisin Carlsberg and Eglin C is distinct from the present complex in that the two types of beta-interactions are not separate. As for the flexibility of the molecules comprising the present complex, the following observations were made by comparing the B-factors for free and complexed SSI and comparing those for free and complexed subtilisin BPN'. The rigidification of the component molecules upon complex formation occurs in a very localized region: in SSI, the "primary" and "secondary" contact regions and the flanking region; in subtilisin BPN', the S1-3 and S4-6 sites and the flanking region.

Bacterial Proteins↗
PubMed1990

Pro-subtilisin E: purification and characterization of its autoprocessing to active subtilisin E in vitro.

The formation of active subtilisin E from pro-subtilisin E requires the removal of the N-terminal pro-sequence of 77 residues. Pro-subtilisin E produced in Escherichia coli using a pINIII-ompA vector was first extracted with 6 M guanidine-HCl and 5 M urea and purified to homogeneity in the presence of 5 M urea. Upon drop dialysis against 0.2 M sodium phosphate buffer (pH 6.2), the purified pro-subtilisin in 5 M urea was processed to active subtilisin of which the N-terminal sequence and migration in SDS-polyacrylamide gel electrophoresis were identical to those of authentic active subtilisin E. This process was found to be very sensitive to the ionic strengths and anions used. Under the optimum conditions (dialysis against 0.5 M (NH4)2SO4 and 1 mM CaCl2 in 10 mM Tris-HCl buffer (pH 7.0) at 4 degrees C for 1 h), approximately 20% of pro-subtilisin E was converted to active subtilisin E. The activation process was not inhibited by Streptomyces subtilisin inhibitor, and pro-subtilisin E in which the active site was mutated (Asp32 to Asn) was unable to be processed under the optimum conditions. These results confirmed the previous hypothesis that the processing of pro-subtilisin occurs by an intramolecular, autoprocessing mechanism.

Ammonium Sulfate↗
PubMed1989

Pro-sequence of subtilisin can guide the refolding of denatured subtilisin in an intermolecular process.

Subtilisin E, an alkaline serine protease consisting of a single polypeptide chain of 275 amino acids is produced from a pre-pro-protein. The pre-sequence functions as the signal peptide for protein secretion across the membrane. Deletion of the pro-sequence yields mature but inactive subtilisin: the 77-amino acid pro-sequence must precede the mature subtilisin to guide the latter into an active conformation. Pro-subtilisin denatured in 6 M guanidine-HCl can be self-processed to the active enzyme intramolecularly, with concomitant cleavage of the pro-sequence, when dialysed against renaturing buffer. We have constructed an active-centre mutant of pro-subtilisin (Asp 32----Asn) which is not processed to active enzyme, unlike the wild-type pro-subtilisin, because intramolecular processing is prevented. Here we report an intermolecular pathway for the refolding of the inactive mature protein to an active enzyme in vitro with the aid of exogenously added pro-sequence. We establish conditions under which the mature inactive form, as well as acid-denatured subtilisins Carlsberg and BPN', can be renatured by the mutant pro-subtilisin.

Enzyme Activation↗
PubMed1979

[Subtilisin 72: a serine protease from Bac. subtilis strain 72 - an enzyme similar to subtilisin Carlsberg].

Subtilisin 72, a serine proteinase secreted by Bac. subtilis strain 72 was purified by covalent chromatography on Sepharose sorbent containing p-(omega-aminomethyl)phenylboronic acid as a ligand. The homogeneity of subtilisin 72 was confirmed by isoelectrofocusing in a thin layer of polyacrylamide gel (pl 8.6). The amino acid composition of this enzyme is different from that of other subtilisins, e. g. subtilisin Carlsberg. The N = terminal amino acid sequence of subtilisin 72 traced up to the 35th residue turned to be the same as that of subtilisin Carlsberg with the exception of the 21st (Tyr) and the 30th (Ile) residues. This very pronounced extent of homology shows that subtilisin 72 is very similar although not identical to subtilisin Carlsberg.

Amino Acid Sequence↗
PubMed1991

Inhibition of subtilisin BPN' by reaction site P1 mutants of Streptomyces subtilisin inhibitor.

It has been shown that the P1 site (the center of the reactive site) of protease inhibitors corresponds to the specificity of the cognate protease, and consequently specificity of Streptomyces subtilisin inhibitor (SSI) can be altered by substitution of a single amino acid at the P1 site. In this paper, to investigate whether similar correlation between inhibitory activity of mutated SSI and substrate preference of protease is observed for subtilisin BPN', which has broad substrate specificity, a complete set of mutants of SSI at the reaction site P1 (position 73) was constructed by cassette and site-directed mutagenesis and their inhibitory activities toward subtilisin BPN' were measured. Mutated SSIs which have a polar (Ser, Thr, Gln, Asn), basic (Lys, Arg), or aromatic amino acid (Tyr, Phe, Trp, His), or Ala or Leu, at the P1 site showed almost the same strong inhibitory activity toward subtilisin as the wild type (Met) SSI. However, the inhibitory activity of SSI variants with an acidic (Glu, Asp), or a beta-branched aliphatic amino acid (Val, Ile), or Gly or Pro, at P1 was decreased. The values of the inhibitor constant (Ki) of mutated SSIs toward subtilisin BPN' were consistent with the substrate preference of subtilisin BPN'. A linear correlation was observed between log(1/Ki) of mutated SSIs and log(1/Km) of synthetic substrates. These results demonstrate that the inhibitory activities of P1 site mutants of SSI are linearly related to the substrate preference of subtilisin BPN', and indicate that the binding mode of the inhibitors with the protease may be similar to that of substrates, as in the case of trypsin and chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1979

The interaction of a tyrosyl residue and carboxyl groups in the specific interaction between Streptomyces subtilisin inhibitor and subtilisin BPN'. A chemical modification study.

An ultraviolet absorption difference spectrum that is typical of a change in ionization state (pKa 9.7 leads to greater than 11.5) of a tyrosyl residue has been observed on the binding between Streptomyces subtilisin inhibitor (SSI) and subtilisin BPN' [EC 3.4.21.14] at alkaline pH, ionic strength 0.1 M, at 25 degrees C (Inouye, K., Tonomura, B., and Hiromi, K., submitted). When the complex of SSI and subtilisin BPN' is formed at an ionic strength of 0.6 M and pH 9.70, the characteristic features of the protonation of a tyrosyl residue in the difference spectrum are diminished. These results suggest that the pKa-shift of a tyrosyl residue observed at alkaline pH and lower ionic strength results from an electrostatic interaction. Nitration of tyrosyl residues of SSI and of subtilisin BPN' was performed with tetranitromethane (TNM). By measurements of the difference spectra observed on the binding of the tyrosyl-residue-nitrated SSI and the native subtilisin BPN', and on the binding of the native SSI and the tyrosyl-residue-nitrated subtilisin BPN' and alkaline pH, the tyrosyl residue in question was shown to be one out of the five tyrosyl residues of pKa 9.7 of the enzyme. This tyrosyl residue was probably either Tyr 217 or Tyr 104 on the basis of the reactivities of tyrosyl residues of the enzyme with TNM and their locations on the enzyme molecule. Carboxyl groups of SSI were modified by covalently binding glycine methyl ester with the aid of water-soluble carbodiimide, in order to neutralize the negative charges on SSI. In the difference spectrum which was observed on the binding of subtilisin BPN' and the 5.3-carboxyl-group-modified SSI at alkaline pH, the characteristic features of the protonation of a tyrosyl residue were essentially lost, and the difference spectrum is rather similar to that observed on the binding of the native SSI and the enzyme at neutral pH. This phenomenon indicates that the pKa of a tyrosyl residue of the enzyme is shifted upwards by interaction with carboxyl group(s) of SSI on the formation of the enzyme-inhibitor complex.

Amino Acids↗
PubMed1987

The high-resolution X-ray crystal structure of the complex formed between subtilisin Carlsberg and eglin c, an elastase inhibitor from the leech Hirudo medicinalis. Structural analysis, subtilisin structure and interface geometry.

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-nm 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 Lys8I (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 beta-sheet flanked by an alpha-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 of two arginine residues which project from the hydrophobic core [Bode, W., Papamokos, E., Musil, D., Seemüller, W. & Fritz, H. (1986) EMBO J. 5, 813-818]. The reactive site loop conformation resembles that found in other 'small' proteinase inhibitors. The scissile peptide bond is not cleaved but its carbonyl group is slightly distorted from planar geometry. Most of the intermolecular contacts are contributed by the nine residues of the reactive-site loop Gly40I-Arg48I.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Determination of the complete amino-acid sequence of subtilisin DY and its comparison with the primary structures of the subtilisins BPN', Carlsberg and amylosacchariticus.

The complete amino-acid sequence of subtilisin DY, an extracellular alkaline proteinase produced by Bacillus subtilis strain DY was determined. This included automated sequence analysis of the whole molecule and its large fragments such as tryptic peptides obtained from the inactivated enzyme, peptides generated by cyanogen bromide, by o-iodosobenzoic acid and by hydroxylamine. The peptides were isolated by gel filtration and by reversed-phase high performance liquid chromatography. The amino-acid sequence of subtilisin DY was determined by overlapping the isolated peptides. It consists of 274 amino-acid residues, like that of subtilisin Carlsberg. By comparison with the structures of the subtilisins Carlsberg, amylosacchariticus and BPN' 32, 80 and 82 amino-acid substitutions were found, which are caused by 37, 102 and 106 nucleotide mutations, respectively. It was found also that 62.5% of the amino-acid residues in the molecules of these four subtilisins are identical with respect to kind and position of the residue, which suggests that these molecules have had a common ancestral precursor. The amino-acid replacement analysis of the four subtilisins leads to the conclusion that they have evolved almost independently.

Amino Acid Sequence↗
PubMed1988

QSAR analysis of the subtilisin hydrolysis of X-phenyl hippurates. II. A study of subtilisin BPN'.

The hydrolysis of 30 substituted phenyl hippurates (X-C6H4OCOCH2NHCOC6H5) by subtilisin BPN' was studied and from the results the following quantitative structure-activity relationship was derived: log 1/Km = 0.39 sigma + 0.16 B5.4 + 0.29 pi'3 + 3.58. In this expression Km is the Michaelis constant, sigma is the Hammett constant, B5.4 is the sterimol steric parameter of X in the 4-position and pi'3 is the hydrophobic parameter for the more hydrophobic of the two possible meta substituents. The other meta substitutent is assigned a pi value of 0. This mathematical model is qualitatively compared with a molecular graphics model constructed from the X-ray crystallographic coordinates of subtilisin BPN'. The results with subtilisin BPN' are compared with our earlier study of similar substrates with Carlsberg subtilisin.

Computer Graphics↗
PubMed1992

Functional analysis of the intramolecular chaperone. Mutational hot spots in the subtilisin pro-peptide and a second-site suppressor mutation within the subtilisin molecule.

The N-terminal pro-peptide of 77 amino acid residues is essential for the folding of subtilisin, an alkaline serine protease from Bacillus subtilis. The synthetic pro-peptide has been shown to be capable of guiding the proper folding of denatured subtilisin to enzymatically active enzyme. Thus the pro-peptide serves as an intramolecular chaperone, which is removed by an autoprocessing reaction after the completion of the folding. With use of localized polymerase chain reaction random mutagenesis a total of 25 amino acid substitution mutations that affected subtilisin activities were isolated. These mutations occurred in a high frequency at the hydrophobic regions of the pro-peptide. For one of the mutations, M(-60)T, a second-site suppressor mutation, S(188)L, was isolated within the mature region. These results suggest that the pro-peptide consists of a few functional regions which interact with specific regions of the mature region of subtilisin during the folding process.

Amino Acid Sequence↗
PubMed1977

The stoichiometry of inhibition and binding of a protein proteinase inhibitor from Streptomyces (Streptomyces subtilisin inhibitor) against subtilisin BPN'1.

The stoichiometry of inhibition and binding of Streptomyces subtilisin inhibitor, a protein proteinase inhibitor produced by Streptomyces albogriseolus S-3253 (Sato, S. and Murao, S. (1973) Agric. Biol. Chem. 37, 1067) against subtilison BPN' [EC 3.4.21.14] was studied. The inhibition of the hydrolysis of p-nitrophenyl acetate by subtilisin BPN' was measured both at the pre-steady state with a stopped-flow apparatus and at the steady state. The stopped-flow study demonstrated the disappearance of the initial burst of the enzyme reaction. The ultraviolet absorption difference spectra observed on mixing the inhibitor and the enzyme suggested changes in the environment of tryptophyl and tyrosyl residues in the proteins. Titration by means either of the degree of inhibition at the steady state or of the magnitude of the ultraviolet difference absorbance revealed that the inhibitor (dimer, MW: 23,000) bound and inhibited two molecules of subtilisin BPN'. The inhibitor constant, Ki, against subtilisin BPN' was estimated to be less than 10(-9)M at pH 8.50. The type of inhibition of this inhibitor is discussed.

Bacterial Proteins↗
PubMed1991

Molecular recognition at the active site of subtilisin BPN': crystallographic studies using genetically engineered proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor).

Unlike trypsin-like serine proteases having only one conspicuous binding pocket in the active site, subtilisin BPN' has two such pockets, the S1 and S4 pockets, which accommodate the P1 and P4 residues of ligands (after Schechter and Berger notation) respectively. Using computer graphics, the geometrical nature of the two pockets was carefully examined and strategies for site-directed mutagenesis studies were set up against a protein SSI (Streptomyces subtilisin inhibitor), which is a strong proteinaceous inhibitor (or a substrate analogue) of subtilisin BPN'. It was decided to convert the P1 residue, methionine 73, into lysine (M73K) with or without additional conversion of the P4 residue, methionine 70, into glycine (M70G). The crystal structures of the two complexes of subtilisin BPN', one with the single mutant SSI (M73K) and the other with the double mutant SSI (M73K, M70G) were solved showing that (i) small 'electrostatic induced-fit movement' occurs in the S1 pocket upon introducing the terminal plus charge of the lysine side chain, and (ii) large 'mechanical induced-fit movement' occurs in the S4 pocket upon reducing the size of the P4 side chain from methionine to glycine. In both (i) and (ii), the induced-fit movement occurred in a concerted fashion involving both the enzyme and 'substrate' amino acid residues. The term 'substrate-assisted stabilization' was coined to stress the cooperative nature of the induced-fit movements.

Bacterial Proteins↗
PubMed1990

Fluorescence labeled and cross-linked subtilisin: kinetic characteristics and binding to Streptomyces subtilisin inhibitor.

In the preceding paper, the preparation of fluorescent cross-linked subtilisin was described. In this paper we present the catalytic and binding properties of the modified enzyme. Kinetic analysis showed that the cross-linked dimeric subtilisin retained both catalytic activity and binding affinity toward synthetic substrates. These kinetic characteristics of the modified enzyme were nearly identical to those of the native enzyme. The modified enzyme also exhibited a specific interaction with Streptomyces subtilisin inhibitor (SSI) with 1 to 1 stoichiometry. The formation of a polymeric complex, which is the expected product with cross-linked subtilisin, was demonstrated.

Bacterial Proteins↗
PubMed1988

Structural comparison of two serine proteinase-protein inhibitor complexes: eglin-c-subtilisin Carlsberg and CI-2-subtilisin Novo.

The crystal structures of the molecular complexes between two serine proteinases and two of their protein inhibitors have been determined: subtilisin Carlsberg with the recombinant form of eglin-c from the leech Hirudo medicinalis and subtilisin Novo with chymotrypsin inhibitor 2 from barley seeds. The structures have been fully refined by restrained-parameter least-squares methods to crystallographic R factors (sigma[[Fo[ - [Fc[[/sigma[Fo[) of 0.136 at 1.8-A resolution and 0.154 at 2.1-A resolution, respectively. The 274 equivalent alpha-carbon atoms of the enzymes superpose with an rms deviation of 0.53 A. Sequence changes between the enzymes result in localized structural adjustments. Functional groups in the active sites superpose with an rms deviation of 0.19 A for 161 equivalent atoms; this close similarity in the conformation of active-site residues provides no obvious reason for known differences in catalytic activity between Carlsberg and Novo. Conformational changes in the active-site region indicate a small induced fit of enzyme and inhibitor. Some conformational differences are observed between equivalent active-site residues of subtilisin Carlsberg and alpha-chymotrypsin. Despite differences in tertiary architecture, most enzyme-substrate (inhibitor) interactions are maintained. Subtilisin Carlsberg has a rare cis-peptide bond preceding Thr211 (Gly211 in Novo). Both enzymes contain tightly bound Ca2+ ions. Site 1 is heptacoordinate with the oxygen atoms at the vertices of a pentagonal bipyramid. Site 2 in Carlsberg is probably occupied by a K+ ion in Novo. Conserved water molecules appear to play important structural roles in the enzyme interior, in the inhibitor beta-sheet, and at the enzyme-inhibitor interface. The 62 equivalent alpha-carbon atoms of the inhibitors superpose with an rms deviation of 1.68 A. Sequence changes result in somewhat different packing of the alpha-helix, beta-sheet, and reactive-site loop relative to each other. Hydrogen bonds and electrostatic interactions supporting the conformation of the reactive-site loop are conserved. The 24 main-chain plus C beta atoms of P4 to P1' overlap with an rms deviation of 0.19 A. Features contributing to the inhibitory nature of eglin-c and CI-2 are discussed.

Amino Acid Sequence↗
PubMed1986

Refined 1.2 A crystal structure of the complex formed between subtilisin Carlsberg and the inhibitor eglin c. Molecular structure of eglin and its detailed interaction with subtilisin.

The crystal structure of the complex formed between eglin c, an elastase inhibitor from the medical leech, and subtilisin Carlsberg has been determined at 1.2 A resolution by a combination of Patterson search methods and isomorphous replacement techniques. The structure has been refined to a crystallographic R-value of 0.18 (8-1.2 A). Eglin consists of a four-stranded beta-sheet with an alpha-helical segment and the protease-binding loop fixed on opposite sides. This loop, which contains the reactive site Leu45I--Asp46I, is mainly held in its conformation by unique electrostatic/hydrogen bond interactions of Thr44I and Asp46I with the side chains of Arg53I and Arg51I which protrude from the hydrophobic core of the molecule. The conformation around the reactive site is similar to that found in other proteinase inhibitors. The nine residues of the binding loop Gly40I--Arg48I are involved in direct contacts with subtilisin. In this interaction, eglin segment Pro42I--Thr44I forms a three-stranded anti-parallel beta-sheet with subtilisin segments Gly100--Gly102 and Ser125--Gly127. The reactive site peptide bond of eglin is intact, and Ser221 OG of the enzyme is 2.81 A apart from the carbonyl carbon.

Amino Acid Sequence↗
PubMed1985

Calorimetric studies of the binding of Streptomyces subtilisin inhibitor to subtilisin of Bacillus subtilis strain N'.

The binding of Streptomyces subtilisin inhibitor (SSI) to subtilisin of Bacillus subtilis strain N' (subtilisin BPN', EC 3.4.21.14) was studied by isothermal calorimetry at pH 7.0 and at various temperatures ranging from 5 to 30 degrees C. Thermodynamic quantities for the binding reaction were derived as a function of temperature by combining the data reported for the dissociation constant with the present calorimetric results. At 25 degrees C, the values are delta G degrees = -57.9 kJ mol-1, delta H = -19.8 kJ mol-1, delta S degree = 0.13 kJ K-1 mol-1, and delta Cp = -1.02 kJ K-1 mol-1. The entropy and the heat capacity changes are discussed in terms of the contributions from the changes in vibrational modes and in hydrophobic interactions.

Bacillus subtilis↗