Alteration of inhibitory properties of Pleurotus ostreatus proteinase A inhibitor 1 by mutation of its C-terminal region

Pleurotus ostreatus proteinase A inhibitor 1 (POIA1), which is composed of 76 residues without disulfide bridges, is a unique inhibitor in that it exhibits sequence similarity to the propeptides of subtilisins. In order to elucidate the inhibitory mechanism of POIA1, we constructed an expression system for a synthetic POIA1 gene. The wild-type POIA1 was found to inhibit subtilisin BPN' with an inhibitor constant (K(i)) of 3.2 x 10(-9) M, but exhibited a time-dependent decrease of inhibitory activity as a consequence of degradation by the protease, showing that the wild-type POIA1 was a temporary inhibitor when subtilisin BPN' was used as a target protease. Since POIA1 shows sequence similarity to the propeptide of subtilisin, which is known to inhibit the protease via its C-terminal region, the C-terminal six residues of POIA1 were replaced with those of the propeptide of subtilisin BPN'. The mutated POIA1 inhibited subtilisin BPN' with a K(i) value of 2.8 x 10(-11) M and did not exhibit time-dependent decrease of inhibitory activity, showing about 100-fold increases in binding affinity for, and resistance to, the protease. These results clearly indicate that the C-terminal region of POIA1 plays an important role in determining the inhibitory activity toward the protease, and that the increase in binding ability to the protease is closely related to resistance to proteolytic degradation. Therefore, the inhibitory properties of POIA1 can be altered by mutation of its C-terminal region.     

Inhibitor-assisted refolding of protease: a protease inhibitor as an intramolecular chaperone

Pleurotus ostrearus proteinase A inhibitor 1 (POIA1), which was discovered as a protease inhibitor, is unique in that it shows sequence homology to the propeptide of subtilisin, which functions as an intramolecular of a cognate protease. In this study, we demonstrate that POIA1 can function as an intramolecular chaperone of subtilisin by in vitro and in vivo experiments. The specific cleavage between POIA1 and the mature region of subtilisin BPN' occurred in a refolding process of a chimera protein, and Bacillus cells transformed with a chimera gene formed a halo on a skim milk plate. The mutational analyses of POIA1 in the chimera protein suggested that the tertiary structure of POIA1 is required for such a function, and that an increase in its ability to bind to subtilisin BPN' makes POIA1 a more effective intramolecular chaperone.     

Requirement for hydrophobic Phe residues in Pleurotus ostreatus proteinase A inhibitor 1 for stable inhibition

Pleurotus ostreatus proteinase A inhibitor 1 (POIA1) has been shown to be unique among the various serine protease inhibitors in that its C-terminal region appears to be the reactive site responsible for its inhibitory action toward proteases. To investigate in more detail the mechanism of inhibition by POIA1, we have been studying its structural requirements for stable inhibition of proteases. In this study, we focused on hydrophobic Phe residues, which are generally located in the interior of protein molecules. A Phe-->Ala replacement at position 44 or 56 was introduced into a 'parent' mutant of POIA1 that had been converted into a strong and resistant inhibitor of subtilisin BPN' by replacement of its six C-terminal residues with those of the propeptide of subtilisin BPN' and the effects on inhibitory properties and structural stability were examined. Both of the mutated POIA1 molecules not only were found to exhibit decreased ability to bind to subtilisin BPN' (80-fold for the F44A mutant and 13-fold for the F56A mutant), but were also converted to temporary inhibitors that were degraded by the protease. The structural stability of the mutated POIA1 was also lowered, as shown by a 13 degrees C decrease in melting temperature for the F56A mutant. In particular, the F44A mutant was found to lose its tertiary structure, as judged from the circular dichroism spectrum, demonstrating that Phe44 is a strict requirement for structural formation by the POIA1 molecule. These results clearly indicate that stabilization of POIA1 by hydrophobic residues in its molecular interior is required for stable inhibition of the protease. This requirement for a stable tertiary structure is shared with other serine protease inhibitors, but other structural requirements seem to differ, in that strong binding with the protease is required for POIA1 whereas conformational rigidity around the reactive site is essential for many other protease inhibitors.     

Structure of POIA1, a homologous protein to the propeptide of subtilisin: implication for protein foldability and the function as an intramolecular chaperone

Solution structure of POIA1 (Pleurotus ostreatus proteinase A inhibitor 1), which functions as an intramolecular chaperone and as an inhibitor to subtilisin, was determined. By making use of the fact that POIA1 is the only structured protein that shows homology to the propeptide of subtilisin, which is unstructured by itself, foldability of this protein was elucidated. It became clear that the evolutionarily conserved residues play two important roles, one for the maintenance of its own structure, and the other for the interaction with subtilisin. Structural softness and mutational tolerance contained in the POIA1 structure makes it an ideal material for designing a foldable protein.     

Involvement of the C-terminal region of yeast proteinase B inhibitor 2 in its inhibitory action

Yeast proteinase B inhibitor 2 (YIB2), which is composed of 74 amino acid residues, is an unusual serine protease inhibitor, since it lacks disulfide bonds. To identify its reactive site for proteases, we constructed an expression system for a synthetic YIB2 gene and then attempted to change the inhibitory properties of YIB2 by amino acid replacements. The purified wild-type YIB2 inhibited the activity of subtilisin BPN', a protein homologous to yeast proteinase B, although its binding ability was not strong, and a time-dependent decrease in its inhibitory activity was observed, demonstrating that wild-type YIB2 behaves as a temporary inhibitor when subtilisin BPN' is the target protease. Since YIB2 exhibits sequence homology to the propeptide of subtilisin, which inhibits a cognate protease using its C-terminal region, we replaced the six C-termi nal residues of YIB2 with those of the propeptide of subtilisin BPN' to make the mutant YIB2m1. This mutant exhibited markedly increased inhibitory activity toward subtilisin BPN' without a time-dependent decrease in its inhibitory activity. Replacement of only the C-terminal Asn of YIB2 by Tyr, or deletion of the C-terminal Tyr of YIB2m1, inhibited subtilisin, but the ability of these mutants to bind subtilisin and their resistance to proteolytic attack were weaker than those of YIB2m1, indicating that the C-terminal residue contributes to the interaction with the protease to a greater extent than the preceding five residues and that the resistance of YIB2 to proteolyic attack is closely related to its ability to bind a protease. These results demonstrate that YIB2 is a unique protease inhibitor that involves its C-terminal region in the interaction with the protease.     

The refined crystal structure of subtilisin Carlsberg at 2.5 A resolution

We report here the X-ray crystal structure of native subtilisin Carlsberg, solved at 2.5 A resolution by molecular replacement and refined by restrained least squares to a crystallographic residual (Formula see text): of 0.206. we compare this structure to the crystal structure of subtilisin BPN'. We find that, despite 82 amino acid substitutions and one deletion in subtilisin Carlsberg relative to subtilisin BPN', the structures of these enzymes are remarkably similar. We calculate an r.m.s. difference between equivalent alpha-carbon positions in subtilisin Carlsberg and subtilisin BPN' of only 0.55 A. This confirms previous reports of extensive structural homology between these two subtilisins based on X-ray crystal structures of the complex of eglin-c with subtilisin Carlsberg [McPhalen, C.A., Schnebli, H.P. and James, M.N.G. (1985) FEBS Lett., 188, 55; Bode, W., Papamokos, E. and Musil, D. (1987) Eur. J. Biochem., 166, 673-692]. In addition, we find that the native active sites of subtilisins Carlsberg and BPN' are virtually identical. While conservative substitutions at residues 217 and 156 may have subtle effects on the environments of substrate-binding sites S1' and S1 respectively, we find no obvious structural correlate for reports that subtilisins Carlsberg and BPN' differ in their recognition of model substrates. In particular, we find no evidence that the hydrophobic binding pocket S1 in subtilisin Carlsberg is 'deeper', 'narrower' or 'less polar' than the corresponding binding site in subtilisin BPN'.     

Accelerated refolding of subtilisin BPN' by tertiary-structure-forming mutants of its propeptide

The propeptide of subtilisin BPN', which functions as an intramolecular chaperone and a temporary inhibitor of subtilisin, is unique in that it acquires its three-dimensional structure by formation of a complex with the cognate protease. We previously showed that the successive amino acid replacements Ala47-->Phe, Gly13-->Ile, and Val65-->Ile in the propeptide to increase its hydrophobicity resulted in formation of a tertiary structure, accompanied by increased ability to bind to the protease and increased resistance to proteolysis. In this study, we examined the effects of these tertiary-structure-forming mutations on the intramolecular chaperone activity of the propeptide. The successive amino acid replacements mentioned above were introduced into pro-subtilisin*, possessing a Ser221-->Ala mutation in the catalytic residue. Refolding experiments were started by rapid dilution of the denatured pro-subtilisin*, and formation of tertiary structure in subtilisin was monitored kinetically by increase in tryptophan fluorescence. The wild-type pro-subtilisin* was found to refold with a rate constant of 4.8 x 10(-3) s(-1) in the equation describing an intramolecular process. The Ala47-->Phe replacement in the propeptide resulted in a 1.2-fold increase in the rate constant of subtilisin refolding. When the additional replacement Gly13-->Ile was introduced, refolding of subtilisin was substantially accelerated, and its kinetics could be fitted to a double exponential process composed of a fast phase with a rate constant of 2.1 x 10(-2) s(-1) and a slow phase with a rate constant of 4.5 x 10(-3) s(-1). The rate constant of the fast phase was increased slightly by a further replacement, Val65-->Ile. Since the slow phase is considered to correspond to proline isomerization, we concluded that tertiary-structure-forming mutations in the propeptide produce positive effects on its intramolecular chaperone activity through acceleration of the propeptide-induced formation of the tertiary structure of subtilisin BPN'.     

Engineering thermostability in subtilisin BPN' by in vitro mutagenesis

A procedure has been developed for the isolation and identification of mutants of the bacterial serine protease, subtilisin, which exhibit enhanced thermostability. The cloned subtilisin BPN' gene from Bacillus amyloliquefaciens was treated with a variety of chemical mutagens to introduce random mutations in the coding sequence. Strains containing the cloned, mutagenized subtilisin gene which produced subtilisin with enhanced thermostability were selected by a simple plate assay procedure, which screens for esterase activity on nitrocellulose filters after preincubation at elevated temperatures. The identification and characterization of eight different stabilizing mutations are described. Several mutants containing various combinations of these stabilizing mutations were constructed by oligonucleotide-directed mutagenesis. Combining independent, stabilizing mutations in the same subtilisin molecule has resulted in an approximate multiplicative decrease in the rate of thermal inactivation. In this way, a variant of subtilisin has been constructed which is about 12-fold more stable than wild-type subtilisin, with no radical changes in the tertiary protein structure but rather minor, independent alterations in amino acid sequence. The ultimate goal in these studies is to be able to accurately predict where stabilizing changes can be made in a protein.     

Protein engineering of disulfide bonds in subtilisin BPN'

Five single-disulfide mutants were studied in subtilisin BPN', a cysteine-free, secreted serine protease from Bacillus amyloliquefaciens. The disulfides were engineered between residues 26-232, 29-119, 36-210, 41-80, and 148-243. These bonds connected a variety of secondary structural elements, located in buried or exposed positions at least 10 A from the catalytic Ser-221, and linked residues that were separated by 39 up to 206 amino acids. All disulfide bonds formed in the enzyme when the expressed protein was secreted from Bacillus subtilis, and the disulfides had only minor effects on the enzyme kinetics. Although these disulfide bonds varied by over 50-fold in their equilibrium constants for reduction with dithiothreitol, there was no correlation between the strength of the disulfide bond and the stability it imparted to the enzyme to irreversible inactivation. In some cases, the disulfide-bonded protein was stabilized greatly relative to its reduced counterpart. However, no disulfide mutant was substantially more stable than wild-type subtilisin BPN'. Some of these results can be rationalized by destabilizing effects of the cysteine mutations that disrupt interactions present in the folded enzyme structure. It is also possible that the rate of irreversible inactivation depends upon the kinetics and not the thermodynamics of unfolding and so the entropically stabilizing effect expected from a disulfide bond may not apply.     

Incorporation of a stabilizing Ca(2+)-binding loop into subtilisin BPN'.

A rational approach was taken to improve the stability of subtilisin BPN' to autoproteolysis. Two sites of autoproteolysis were identified by isolation of early autolysis products and amino-terminal sequence analysis. These studies showed that subtilisin rapidly cleaves Ala48-Ser49 and Ser163-Thr164 peptide bonds at elevated temperatures. These two sites appear in regions of high mobility as estimated from crystallographic B-factors and are in extended surface loops. To improve the resistance to thermal-induced autolysis, we replaced sequences around these two sites with sequences derived from a thermophilic homologue of subtilisin, thermitase. Thermitase contains a Ca(2+)-binding site in the region surrounding Ser49. When the Ca(2+)-binding segment of thermitase corresponding to residues 45-63 of subtilisin BPN' was installed into subtilisin BPN', the chimeric protein gained the ability to bind another Ca2+ with moderate affinity (Kd approximately 100 microM). This enzyme had the same kcat as wild-type, had a KM value 8-fold larger than wild-type, and was slightly less stable to thermal inactivation in EDTA. However, in 10 mM CaCl2, the mutant subtilisin BPN' was 10-fold more stable to irreversible inactivation at 60 degrees C than wild-type subtilisin BPN' as measured by residual activity against the substrate sAAPF-pna. Next, mutations and deletions derived from thermitase were introduced near the second autolysis loop in subtilisin BPN' (residues 158-165). However, all of these mutants were less stable than wild-type subtilisin. Thus, some (but not all) mutations derived from a thermophilic homologue near sites of autolysis can be stabilizing to a mesophilic protease.     

Homology modelling and protein engineering strategy of subtilases, the family of subtilisin-like serine proteinases.

Subtilases are members of the family of subtilisin-like serine proteases. Presently, greater than 50 subtilases are known, greater than 40 of which with their complete amino acid sequences. We have compared these sequences and the available three-dimensional structures (subtilisin BPN', subtilisin Carlsberg, thermitase and proteinase K). The mature enzymes contain up to 1775 residues, with N-terminal catalytic domains ranging from 268 to 511 residues, and signal and/or activation-peptides ranging from 27 to 280 residues. Several members contain C-terminal extensions, relative to the subtilisins, which display additional properties such as sequence repeats, processing sites and membrane anchor segments. Multiple sequence alignment of the N-terminal catalytic domains allows the definition of two main classes of subtilases. A structurally conserved framework of 191 core residues has been defined from a comparison of the four known three-dimensional structures. Eighteen of these core residues are highly conserved, nine of which are glycines. While the alpha-helix and beta-sheet secondary structure elements show considerable sequence homology, this is less so for peptide loops that connect the core secondary structure elements. These loops can vary in length by greater than 150 residues. While the core three-dimensional structure is conserved, insertions and deletions are preferentially confined to surface loops. From the known three-dimensional structures various predictions are made for the other subtilases concerning essential conserved residues, allowable amino acid substitutions, disulphide bonds, Ca(2+)-binding sites, substrate-binding site residues, ionic and aromatic interactions, proteolytically susceptible surface loops, etc. These predictions form a basis for protein engineering of members of the subtilase family, for which no three-dimensional structure is known.     

Stabilizing the subtilisin BPN' pro-domain by phage display selection: How restrictive is the amino acid code for maximum protein stability?

We have devised a procedure using monovalent phage display to select for stable mutants in the pro-domain of the serine protease, subtilisin BPN'. In complex with subtilisin, the pro-domain assumes a compact structure with a four-stranded antiparallel beta-sheet and two three-turn alpha-helices. When isolated, however, the pro-domain is 97% unfolded. These experiments use combinatorial mutagenesis to select for stabilizing amino acid combinations at a particular structural locus and determine how many combinations are close to the maximum protein stability. The selection for stability is based on the fact that the independent stability of the pro-domain is very low and that binding to subtilisin is thermodynamically linked to folding. Two libraries of mutant pro-domains were constructed and analyzed to determine how many combinations of amino acids at a particular structural locus result in the maximum stability. A library comprises all combinations of four amino acids at a structural locus. Previous studies using combinatorial genetics have shown that many different combinations of amino acids can be accommodated in a selected locus without destroying function. The present results indicate that the number of sequence combinations at a structural locus, which are close to the maximum stability, is small. The most striking example is a selection at an interior locus of the pro-domain. After two rounds of phagemid selection, one amino acid combination is found in 40% of sequenced mutants. The most frequently selected mutant has a deltaG(unfolding) = 4 kcal/mol at 25 degrees C, an increase of 6 kcal/mol relative to the naturally occurring sequence. Some implications of these results on the amount of sequence information needed to specify a unique tertiary fold are discussed. Apart from possible implications on the folding code, the phage display selection described here should be useful in optimizing the stability of other proteins, which can be displayed on the phage surface.     

A novel double-headed proteinaceous inhibitor for metalloproteinase and serine proteinase

A novel proteinaceous inhibitor for the metalloproteinase of Streptomyces caespitosus has been isolated from the culture supernatant of Streptomyces sp. I-355. It was named ScNPI (Streptomyces caespitosus neutral proteinase inhibitor). ScNPI exhibited strong inhibitory activity toward ScNP with a K(i) value of 1.6 nm. In addition, ScNPI was capable of inhibiting subtilisin BPN' (K(i) = 1.4 nm) (EC ). The scnpi gene consists of two regions, a signal peptide (28 amino acid residues) and a mature region (113 amino acid residues, M(r) = 11,857). The deduced amino acid sequence of scnpi showed high similarity to those of Streptomyces subtilisin inhibitor (SSI) and its homologues. The reactive site of ScNPI for inhibition of subtilisin BPN' was identified to be Met(71)-Tyr(72) bond by specific cleavage. To identify the reactive site for ScNP, Tyr(33) and Tyr(72), which are not conserved among other SSI family inhibitors but are preferable amino acid residues for ScNP, were replaced separately by Ala. The Y33A mutant retained inhibitory activity toward subtilisin BPN' but did not show any inhibitory activity toward ScNP. Moreover, a dimer of ternary complexes among ScNPI, ScNP, and subtilisin BPN' was formed to give the 2:2:2 stoichiometry. These results strongly indicate that ScNPI is a double-headed inhibitor that has individual reactive sites for ScNP and subtilisin BPN'.     

Folding pathway mediated by an intramolecular chaperone. A functional peptide chaperone designed using sequence databases

Catalytic domains of several prokaryotic and eukaryotic protease families require dedicated N-terminal propeptide domains or "intramolecular chaperones" to facilitate correct folding. Amino acid sequence analysis of these families establishes three important characteristics: (i) propeptides are almost always less conserved than their cognate catalytic domains, (ii) they contain a large number of charged amino acids, and (iii) propeptides within different protease families display insignificant sequence similarity. The implications of these findings are, however, unclear. In this study, we have used subtilisin as our model to redesign a peptide chaperone using information databases. Our goal was to establish the minimum sequence requirements for a functional subtilisin propeptide, because such information could facilitate subsequent design of tailor-made chaperones. A decision-based computer algorithm that maintained conserved residues but varied all non-conserved residues from a multiple protein sequence alignment was developed and utilized to design a novel peptide sequence (ProD). Interestingly, despite a difference of 5 pH units between their isoelectric points and despite displaying only 16% sequence identity with the wild-type propeptide (ProWT), ProD chaperones folding and functions as a potent subtilisin inhibitor. The computed secondary structures and hydrophobic patterns within these two propeptides are similar. However, unlike ProWT, ProD adopts a well defined alpha-beta conformation as an isolated peptide and forms a stoichiometric complex with mature subtilisin. The CD spectra of this complex is similar to ProWT.subtilisin. Our results establish that despite low sequence identity and dramatically different charge distribution, both propeptides adopt similar structural scaffolds. Hence, conserved scaffolds and hydrophobic patterns, but not absolute charge, dictate propeptide function.     

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)     

Molecular cloning of the structural gene for alkaline elastase YaB, a new subtilisin produced by an alkalophilic Bacillus strain.

Alkaline elastase YaB is an extracellular serine protease of the alkalophilic Bacillus strain YaB. We cloned the structural gene, ale, and determined the nucleotide sequence. The mature enzyme (268 amino acids) was preceded by a putative signal sequence and a prosequence (27 and 83 amino acids, respectively). The mature enzyme was 55% homologous to subtilisin BPN'. Almost all the positively charged residues are predicted to be on the surface of the molecule, which would facilitate binding to elastin. The P1 substrate site-related sequences differed between alkaline elastase YaB and subtilisin BPN'.     

Assignment of histidine resonances in the 1H NMR (500 MHz) spectrum of subtilisin BPN' using site-directed mutagenesis

A spin-echo pulse sequence has been used to resolve the six histidine C-2H protons in the 500-MHz NMR spectrum of subtilisin BPN'. Five of these residues have been substituted by site-directed mutagenesis, and this has enabled a complete assignment of these protons to be obtained. Analysis of the pH titration curves of these signals has provided microscopic pKas for the six histidines in this enzyme. The pKas of the histidine residues in subtilisin BPN' have been compared with the values obtained for the histidines in the homologous enzyme from Bacillus licheniformis (subtilisin Carlsberg). Four of the five conserved histidines titrate with essentially identical pKa's in the two enzymes. It therefore appears that the assignments made for these residues in subtilisin BPN' can be transferred to subtilisin Carlsberg. On the basis of these assignments, the one histidine that titrates with a substantially different pKa in the two enzymes can be assigned to histidine-238. This difference in pKa has been attributed to a Trp to Lys substitution at position 241 in subtilisin Carlsberg.     

Enzyme-substrate interactions in the hydrolysis of peptide substrates by thermitase, subtilisin BPN', and proteinase K

Peptide substrates of the general structure acetyl-Alan (n = 2-5), acetyl-Pro-Ala-Pro-Phe-Alan-NH2 (n = 0-3), and acetyl-Pro-Ala-Pro-Phe-AA-NH2 (AA = various amino acids) were synthesized and used to investigate the enzyme-substrate interactions of the microbial serine proteases thermitase, subtilisin BPN', and proteinase K on the C-terminal side of the scissile bond. The elongation of the substrate peptide chain up to the second amino acid on the C-terminal side (P'2) enhances the hydrolysis rate of thermitase and subtilisin BPN', whereas for proteinase K an additional interaction with the third amino acid (P'3) is possible. The enzyme subsite S'1 specificity of the proteases investigated is very similar. With respect to kcat/Km values small amino acid residues such as Ala and Gly are favored in this position. Bulky residues such as Phe and Leu were hydrolyzed to a lower extent. Proline in P'1 abolishes the hydrolysis of the substrates. Enzyme-substrate interactions on the C-terminal side of the scissile bond appear to affect kcat more than Km for all three enzymes.     

The role of tryptophan residues in the autoprocessing of prosubtilisin E

Subtilisin E, a serine protease from Bacillus subtilis, requires an N-terminal propeptide for its correct folding. The propeptide is autocleaved and digested by the subtilisin domain upon proper folding. Here we investigated the individual roles of the three Trp residues within the subtilisin domain (Trp106, Trp113 and Trp241) on propeptide processing, enzymatic activity and stability of subtilisin. When the propeptide processing was examined by SDS-PAGE after refolding by rapid dilution, the mutation at either position Trp106 or Trp113 was found to significantly delay the propeptide processing, while the mutation at Trp241 had no effect. Far-UV circular dichroism (CD) spectra of the mutants revealed that the mutations at the three positions did not affect appreciably the alpha-helix content of subtilisin. Secondary structure thermal unfolding monitored by CD spectroscopy revealed that none of the tryptophan residues had any significant effect on the stability of mature subtilisin. The enzymatic activity measurements showed that only Trp106 plays a major role in the enzymatic activity of subtilisin E. These results demonstrate that both Trp106 and Trp113 play a specific role in propeptide processing and enzymatic activity, while Trp241 plays no considerable role on any of these activities.     

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.