Streptomyces Griseus Protease B Research Articles

Additivity-based design of the strongest possible turkey ovomucoid third domain inhibitors for porcine pancreatic elastase (PPE) and Streptomyces griseus protease B (SGPB)

We describe here successful designs of strong inhibitors for porcine pancreatic elastase (PPE) and Streptomyces griseus protease B (SGPB). For each enzyme two inhibitor variants were designed. In one, the reactive site residue (position 18) was retained and the best residues were substituted at contact positions 13, 14, and 15. In the other variant the best residues were substituted at all contact positions except the reactive site where a Gly was substituted. The four designed variants were: for PPE, T13E14Y15-OMTKY3 and T13E14Y15G18M21P32V36-OMTKY3, and for SGPB, S13D14Y15-OMTKY3 and S13D14Y15G18I19K21-OMTKY3. The free energies of association (ΔG0) of expressed variants have been measured with the proteases for which they were designed as well as with five other serine proteases and the results are discussed.

Functional modulation of a protein folding landscape via side-chain distortion

Ultrahigh-resolution (< 1.0 Å) structures have revealed unprecedented and unexpected details of molecular geometry, such as the deformation of aromatic rings from planarity. However, the functional utility of such energetically costly strain is unknown. The 0.83 Å structure of α-lytic protease (αLP) indicated that residues surrounding a conserved Phe side-chain dictate a rotamer which results in a ~6° distortion along the side-chain, estimated to cost 4 kcal/mol. By contrast, in the closely related protease Streptomyces griseus Protease B (SGPB), the equivalent Phe adopts a different rotamer and is undistorted. Here, we report that the αLP Phe side-chain distortion is both functional and conserved in proteases with large pro regions. Sequence analysis of the αLP serine protease family reveals a bifurcation separating those sequences expected to induce distortion and those that would not, which correlates with the extent of kinetic stability. Structural and folding kinetics analyses of family members suggest that distortion of this side-chain plays a role in increasing kinetic stability within the αLP family members that use a large Pro region. Additionally, structural and kinetic folding studies of mutants demonstrate that strain alters the folding free energy landscape by destabilizing the transition state (TS) relative to the native state (N). Although side-chain distortion comes at a cost of foldability, it suppresses the rate of unfolding, thereby enhancing kinetic stability and increasing protein longevity under harsh extracellular conditions. This ability of a structural distortion to enhance function is unlikely to be unique to αLP family members and may be relevant in other proteins exhibiting side-chain distortions.

Cleavage of peptide bonds bearing ionizable amino acids at P 1 by serine proteases with hydrophobic S 1 pocket

Enzymatic hydrolysis of the synthetic substrate succinyl-Ala-Ala-Pro-Xxx-pNA (where Xxx = Leu, Asp or Lys) catalyzed by bovine chymotrypsin (CHYM) or Streptomyces griseus protease B (SGPB) has been studied at different pH values in the pH range 3–11. The pH optima for substrates having Leu, Asp, and Lys have been found to be 7.5–8.0, 5.5–6.0, and ∼10, respectively. At the normally reported pH optimum (pH 7–8) of CHYM and SGPB, the substrate with Leu at the reactive site is more than 25,000-fold more reactive than that with Asp. However, when fully protonated, Asp is nearly as good a substrate as Leu. The p K values of the side chains of Asp and Lys in the hydrophobic S 1 pocket of CHYM and SGPB have been calculated from pH-dependent hydrolysis data and have been found to be about 9 for Asp and 7.4 and 9.7 for Lys for CHYM and SGPB, respectively. The results presented in this communication suggest a possible application of CHYM like enzymes in cleaving peptide bonds contributed by acidic amino acids between pH 5 and 6.

Structural and Functional Study of an Anemonia Elastase Inhibitor, a “Nonclassical” Kazal-Type Inhibitor fromAnemonia sulcata

Anemonia elastase inhibitor (AEI) is a "nonclassical" Kazal-type elastase inhibitor from Anemonia sulcata. Unlike many nonclassical inhibitors, AEI does not have a cystine-stabilized alpha-helical (CSH) motif in the sequence. We chemically synthesized AEI and determined its three-dimensional solution structure by two-dimensional NMR spectroscopy. The resulting structure of AEI was characterized by a central alpha-helix and a three-stranded antiparallel beta-sheet of a typical Kazal-type inhibitor such as silver pheasant ovomucoid third domain (OMSVP3), even though the first and fifth half-cystine residues forming a disulfide bond in AEI are shifted both toward the C-terminus in comparison with those of OMSVP3. Synthesized AEI exhibited unexpected strong inhibition toward Streptomyces griseus protease B (SGPB). Our previous study [Hemmi, H., et al. (2003) Biochemistry 42, 2524-2534] demonstrated that the site-specific introduction of the engineered disulfide bond into the OMSVP3 molecule to form the CSH motif could produce an inhibitor with a narrower specificity. Thus, the CSH motif-containing derivative of AEI (AEI analogue) was chemically synthesized when a Cys(4)-Cys(34) bond was changed to a Cys(6)-Cys(31) bond. The AEI analogue scarcely inhibited porcine pancreatic elastase (PPE), even though it exhibited almost the same potent inhibitory activity toward SGPB. For the molecular scaffold, essentially no structural difference was detected between the two, but the N-terminal loop from Pro(5) to Ile(7) near the putative reactive site (Met(10)-Gln(11)) in the analogue moved by 3.7 A toward the central helix to form the introduced Cys(6)-Cys(31) bond. Such a conformational change in the restricted region correlates with the specificity change of the inhibitor.

Comprehensive Analysis of Protein Folding Activation Thermodynamics Reveals a Universal Behavior Violated by Kinetically Stable Proteases

Alpha-lytic protease (alpha LP) and Streptomyces griseus protease B (SGPB) are two extracellular serine proteases whose folding is absolutely dependent on the existence of their companion pro regions. Moreover, the native states of these proteins are, at best, marginally stable, with the apparent stability resulting from being kinetically trapped in the native state by large barriers to unfolding. Here, in an effort to understand the physical properties that distinguish kinetically and thermodynamically stable proteins, we study the temperature-dependences of the folding and unfolding kinetics of alpha LP and SGPB without their pro regions, and compare their behavior to a comprehensive set of other proteins. For the folding activation thermodynamics, we find some remarkable universal behaviors in the thermodynamically stable proteins that are violated dramatically by alpha LP. Despite significant variations in deltaC(P,F)++, the maximal folding speed occurs within the narrow biological temperature range for all proteins, except for alpha LP, with its maximal folding speed shifted lower by 200 K. This implies evolutionary pressures on folding speed for typical proteins, but not for alpha LP. In addition, the folding free energy barrier in the biological temperature range for most proteins is predominantly enthalpic, but purely entropic for alpha LP. The unfolding of alpha LP and SGPB is distinguished by three properties: a remarkably large deltaC(P,U)++, a very high deltaG(U)++, and a maximum deltaG(u)++ at the optimal growth temperature for the organism. While other proteins display each of these traits to some approximation, the simultaneous optimization of all three occurs only in the kinetically stable proteins, and appears to be required to maximize their unfolding cooperativity, by suppressing local unfolding events, and slowing the rate of global unfolding. Together, these properties extend the lifetime of these enzymes in the highly proteolytic extracellular environment. Attaining such functional properties seems possible only through the gross perturbation of the folding thermodynamics, which in turn has required the co-evolution of pro regions as folding catalysts.

Generation of a thermostable and denaturant-resistant peptide ligase.

The construction and characterization of a novel, thermostable, peptide ligase are described. Three amino acid substitutions were introduced into the secreted bacterial protease Streptomyces griseus protease B (SGPB). Mutations were chosen on the basis of two separate observations: (i) that a single substitution of the nucleophilic serine (S195A) created an enzyme with significant peptide-ligation activity, albeit greatly reduced stability [(2000) Chem. Biol. 7, 163], and (ii) that a pair of substitutions in the substrate-binding pocket (T213L and F228H) greatly increased the thermostability of the wild-type enzyme [(1996) J. Mol. Biol. 257, 233]. The triple mutant, named streptoligase, was found to catalyze peptide ligation (aminolysis of both a thiobenzyl ester and a p-nitroanilide-activated peptide) efficiently in nondenaturing and denaturing conditions including SDS (0.5% w/v) and guanidine hydrochloride (4.0 M). Moreover, streptoligase exhibited a half-live for unfolding of 16.3 min at 55 degrees C in the absence of stabilizing substrates. The fraction of the streptoligase-catalyzed reaction that gave coupled product with the acceptor peptide FAASR-NH(2) was greater for the p-nitroanilide donor (Sc-AAPF-pNA) than for the benzyl thioester substrate (Sc-AAPF-SBn). These observations are consistent with ligation proceeding through an acyl-enzyme intermediate involving histidine-57. In the case of the thioester donor the triple mutant promotes the direct attack of water on the thioester carbonyl carbon, in addition to hydrolysis occurring at the stage of the acyl-enzyme intermediate. The strategy of multiple point mutations outlined in this study may provide a general means of converting enzymes with chymotrypsin-like protein folds into peptide ligases.

The folding landscape of Streptomyces griseus protease B reveals the energetic costs and benefits associated with evolving kinetic stability.

Like most extracellular bacterial proteases, Streptomyces griseus protease B (SGPB) and alpha-lytic protease (alphaLP) are synthesized with covalently attached pro regions necessary for their folding. In this article, we characterize the folding free energy landscape of SGPB and compare it to the folding landscapes of alphaLP and trypsin, a mammalian homolog that folds independently of its zymogen peptide. In contrast to the thermodynamically stable native state of trypsin, SGPB and alphaLP fold to native states that are thermodynamically marginally stable or unstable, respectively. Instead, their apparent stability arises kinetically, from unfolding free energy barriers that are both large and highly cooperative. The unique unfolding transitions of SGPB and alphaLP extend their functional lifetimes under highly degradatory conditions beyond that seen for trypsin; however, the penalty for evolving kinetic stability is remarkably large in that each factor of 2.4-8 in protease resistance is accompanied by a cost of ~10(5) in the spontaneous folding rate and ~5-9 kcal/mole in thermodynamic stability. These penalties have been overcome by the coevolution of increasingly effective pro regions to facilitate folding. Despite these costs, kinetic stability appears to be a potent mechanism for developing native-state properties that maximize protease longevity.

Contribution of peptide bonds to inhibitor-protease binding: crystal structures of the turkey ovomucoid third domain backbone variants OMTKY3-Pro18I and OMTKY3-Ψ[COO]-Leu18I in complex with Streptomyces griseus proteinase B (SGPB) and the structure of the free inhibitor, OMTKY3-Ψ[CH 2NH 2+]-Asp19I

X-ray crystallography has been used to determine the 3D structures of two complexes between Streptomyces griseus proteinase B (SGPB), a bacterial serine proteinase, and backbone variants of turkey ovomucoid third domain (OMTKY3). The natural P 1 residue (Leu18I) has been substituted by a proline residue (OMTKY3-Pro18I) and in the second variant, the peptide bond between Thr17I and Leu18I was replaced by an ester bond (OMTKY3-Ψ[COO]-Leu18I). Both variants lack the P 1 NH group that donates a bifurcated hydrogen bond to the carbonyl O of Ser214 and O γ of the catalytic Ser195, one of the common interactions between serine proteinases and their canonical inhibitors. The SGPB:OMTKY3-Pro18I complex has many structural differences in the vicinity of the S 1 pocket when compared with the previously determined structure of SGPB:OMTKY3-Leu18I. The result is a huge difference in the Δ G° of binding (8.3 kcal/mol), only part of which can be attributed to the missing hydrogen bond. In contrast, very little structural difference exists between the complexes of SGPB:OMTKY3-Ψ[COO]-Leu18I and SGPB:OMTKY3-Leu18I, aside from an ester O replacing the P 1 NH group. Therefore, the difference in Δ G°, 1.5 kcal/mol as calculated from the measured equilibrium association constants, can be attributed to the contribution of the P 1 NH hydrogen bond toward binding. A crystal structure of OMTKY3 having a reduced peptide bond between P 1 Leu18I and P 1′ Asp19I, (OMTKY3-Ψ[CH 2NH 2 +]-Asp19I) has also been determined by X-ray crystallography. This variant has very weak association equilibrium constants with SGPB and with chymotrypsin. The structure of the free inhibitor suggests that the reduced peptide bond has not introduced any major structural changes in the inhibitor. Therefore, its poor ability to inhibit serine proteinases is likely due to the disruptions of the canonical interactions at the oxyanion hole.

Active-site variants of Streptomyces griseus protease B with peptide-ligation activity

Background: Peptide-ligating technologies facilitate a range of manipulations for the study of protein structure and function that are not possible using conventional genetic or mutagenic methods. To different extents, the currently available enzymatic and nonenzymatic methodologies are synthetically demanding, sequence-dependent and/or sensitive to denaturants. No single coupling method is universally applicable. Accordingly, new strategies for peptide ligation are sought.Results: Site-specific variants (Ser195→Gly, S195G, and Ser195→Ala, S195A) of Streptomyces griseus protease B (SGPB) were generated that efficiently catalyze peptide ligation (i.e., aminolysis of ester-, thioester- and para-nitroanilide-activated peptides). The variants also showed reduced hydrolytic activity relative to the wild-type enzyme. The ratio of aminolysis to hydrolysis was greater for the S195A variant, which was also capable of catalyzing ligation in concentrations of urea as high as 2 M.Conclusions: Mutagenic substitution of the active-site serine residue of SGPB by either glycine or alanine has created a unique class of peptide-ligating catalysts that are useful for coupling relatively stable ester- and para-nitroanilide-activated substrates. Ligation proceeds through an acyl–enzyme intermediate involving His57. Serine to alanine mutations may provide a general strategy for converting proteases with chymotrypsin-like protein folds into peptide-coupling enzymes.

Deleterious effects of beta-branched residues in the S1 specificity pocket of Streptomyces griseus proteinase B (SGPB): crystal structures of the turkey ovomucoid third domain variants Ile18I, Val18I, Thr18I, and Ser18I in complex with SGPB.

Turkey ovomucoid third domain (OMTKY3) is a canonical inhibitor of serine proteinases. Upon complex formation, the inhibitors fully exposed P1 residue becomes fully buried in the preformed cavity of the enzyme. All 20 P1 variants of OMTKY3 have been obtained by recombinant DNA technology and their equilibrium association constants have been measured with six serine proteinases. To rationalize the trends observed in this data set, high resolution crystal structures have been determined for OMTKY3 P1 variants in complex with the bacterial serine proteinase, Streptomyces griseus proteinase B (SGPB). Four high resolution complex structures are being reported in this paper; the three beta-branched variants, Ile18I, Val18I, and Thr18I, determined to 2.1, 1.6, and 1.7 A resolution, respectively, and the structure of the Ser18I variant complex, determined to 1.9 A resolution. Models of the Cys18I, Hse18I, and Ape18I variant complexes are also discussed. The beta-branched side chains are not complementary to the shape of the S1 binding pocket in SGPB, in contrast to that of the wild-type gamma-branched P1 residue for OMTKY3, Leu18I. Chi1 angles of approximately 40 degrees are imposed on the side chains of Ile18I, Val18I, and Thr18I within the S1 pocket. Dihedral angles of +60 degrees, -60 degrees, or 180 degrees are more commonly observed but 40 degrees is not unfavorable for the beta-branched side chains. Thr18I Ogamma1 also forms a hydrogen bond with Ser195 Ogamma in this orientation. The Ser18I side chain adopts two alternate conformations within the S1 pocket of SGPB, suggesting that the side chain is not stable in either conformation.

Computational analysis of the binding of P1 variants of domain 3 of turkey ovomucoid inhibitor to Streptomyces griseus protease B

Binding constants for complexes of variants of the ovomucoid inhibitor domain 3 from turkey (OMTKY3) and Streptomyces griseus protease B (SGPB) have been computed. On the basis of the crystallographically determined structures of the complexes, continuum electrostatic calculations have been carried out to evaluate the electrostatic contribution to the binding energy. The hydrophobic component was computed based on the change in the solvent accessible surface area on complex formation. These two terms were combined linearly and the parameters for the protein dielectric, atomic solvation parameter and a constant term were derived using a multivariate fit to the observed binding energies. The resulting fit shows a high correlation with a multiple coefficient of determination of 0.79. This indicates that 79% of the variation in the observed binding energies is explained by the electrostatic and hydrophobic terms. The analysis results in a protein dielectric of 8.2 and an atomic solvation parameter of 30 cal/mol Å2. As a test, these parameters were used to calculate the binding energies of complexes of chymotrypsin and of leukocyte elastase OMTKY3, as well as three other variants of OMTKY3 bound to SGPB. As these structures were not used for the multivariate fit, they serve as an independent check on the derived parameters. The calculated energies for the three new variants of OMTKY3 are in good agreement with the observed values. However, the binding energies of the other complexes are poorly predicted. This implies that the parameters that were obtained are not transferable. The possible causes for this lack of transferability are discussed.

Selection of Streptomyces griseusProtease B Mutants With Desired Alterations in Primary Specificity Using a Library Screening Strategy

Streptomyces griseusprotease B (SGPB) has primary specificity for large hydrophobic residues. The protease is secreted in a promature form, and autocatalytic removal of the propeptide is essential for activity. We genetically substituted the P1 Leu at the promature junction of SGPB with Phe, Met, or Val and monitored expression levels in Escherichia coli. Substitution with Phe had no effect on active SGPB production; substitution with Met or Val abolished proteolytic activity. An E. coliexpression library containing 29,952 possible SGPB mutants was constructed with variations at seven sites involved in conferring primary specificity. A rapid, visual screening strategy was used to detect active protease secretion. The expression library was screened, in conjunction with the different promature junction sequences, for those variants producing increased proteolytic activity. The sequences of the isolated mutant genes were determined; the substrate specificities and thermostabilities of the corresponding proteases were investigated. Mutants isolated from the screen with the wild-type promature junction exhibited substrate specificities and thermostabilities similar to wild-type. The screen with Phe at the promature junction P1 site resulted in the isolation of mutant proteases with increased thermostabilities (up to an order of magnitude increase in half-life at 55°C), while a protease with broad substrate specificity was isolated from the Val screen. Proteases isolated from the screen with Met at the promature junction P1 site exhibited dramatic increases in activity towards a synthetic substrate with Met at the P1 site. The results suggest that the substrate specificity of recombinant SGPB is constrained by the sequence of the promature junction; active protease production is dependent on the efficiency of the self-processive promature junction cleavage. With an efficient screening strategy, this relationship can be used to isolate catalytically active proteases with desired specificities engineered at the promature junction.

Ionizable P1 Residues in Serine Proteinase Inhibitors Undergo Large pK Shifts on Complex Formation

The burial of charged residues in proteins is rare as it is thermodynamically strongly disfavored. However, in "standard mechanism" protein inhibitors of serine proteinases, the P1 residue, which is highly exposed, becomes buried in the S1 specificity pocket of the enzyme. In many enzymes, such as Streptomyces griseus proteinase B (SGPB) the S1 pocket is hydrophobic. We measured the pH dependence of the association equilibrium constant for the interaction of SGPB with turkey ovomucoid third domain P1 mutants, Glu18 OMTKY3 and His18 OMTKY3. In order to eliminate the effects of other ionizable groups on the enzyme and the inhibitor, we divided these pH dependences by the pH dependence of the association equilibrium constant for the Gln18 OMTKY3 mutant. This yielded for Glu18, pKf (free inhibitor) of 4.46 +/- 0.05 and pKc (complex) of 8.74 +/- 0.06. For His18 the values are pKf 6.63 +/- 0.08 and pKc 4.31 +/- 0.07. At low pH values Glu18 variant is a relatively good inhibitor for SGPB. This may be biologically relevant.

Water molecules participate in proteinase‐inhibitor interactions: Crystal structures of Leu18, Ala18, and Gly18 variants of turkey ovomucoid inhibitor third domain complexed with Streptomyces griseus proteinase B

Crystal structures of the complexes of Streptomyces griseus proteinase B (SGPB) with three P1 variants of turkey ovomucoid inhibitor third domain (OMTKY3), Leu18, Ala18, and Gly18, have been determined and refined to high resolution. Comparisons among these structures and of each with native, uncomplexed SGPB reveal that each complex features a unique solvent structure in the S1 binding pocket. The number and relative positions of water molecules bound in the S1 binding pocket vary according to the size of the side chain of the P1 residue. Water molecules in the S1 binding pocket of SGPB are redistributed in response to the complex formation, probably to optimize hydrogen bonds between the enzyme and the inhibitor. There are extensive water-mediated hydrogen bonds in the interfaces of the complexes. In all complexes, Asn 36 of OMTKY3 participates in forming hydrogen bonds, via water molecules, with residues lining the S1 binding pocket of SGPB. For a homologous series of aliphatic straight side chains, Gly18, Ala18, Abu18, Ape18, and Ahp18 variants, the binding free energy is a linear function of the hydrophobic surface area buried in the interface of the corresponding complexes. The resulting constant of proportionality is 34.1 cal mol-1 A-2. These structures confirm that the binding of OMTKY3 to the preformed S1 pocket in SGPB involves no substantial structural disturbances that commonly occur in the site-directed mutagenesis studies of interior residues in other proteins, thus providing one of the most reliable assessments of the contribution of the hydrophobic effect to protein-complex stability.

Inhibition of Serine Proteinases by Squash Inhibitors

The squash inhibitors of serine proteinases have been discovered as proteins, which inhibit the catalytic activity of bovine trypsin. In this report we show, that three human enzymes of trypsin-like specificity - i.e. plasmin, plasma kallikrein and thrombin - are also inhibited by squash inhibitors. Moreover, rather strong inhibition was demonstrated for human cathepsin G. Lower association constants were found for Streptomyces griseus proteinase B (SGPB) and subtilisin BPN'. No association was detected for bovine chymotrypsin, even at millimolar concentrations of the inhibitors. Porcine pancreatic elastase showed extremely weak inhibition by squash inhibitors. Most of the enzymes examined did not exhibit a clear discrimination between P1 Arg and P1 Lys inhibitors. However, human plasma kallikrein and human thrombin formed much stronger complexes with CMTI I (P1-Arg) than with CPTI II (P1-Lys).

Structure of the complex of Streptomyces griseus proteinase B and polypeptide chymotrypsin inhibitor-1 from Russet Burbank potato tubers at 2.1 Å resolution

A low molecular weight protein inhibitor of serine proteinases from Russet Burbank potato tubers, polypeptide chymotrypsin inhibitor-1 (PCI-1), has been crystallized in complex with Streptomyces griseus proteinase B (SGPB). The three-dimensional structure of the complex has been solved at 2.1 Å resolution by the molecular replacement method and has been refined to a final R-factor (= Σ∥F o¦−¦F c∥ Σ¦F o¦ ) of 0.142 (8.0 to 2.1 Å resolution data). The reactive site bond of PCI-1 (Leu38I to Asn39I) is intact in the complex, and there is no significant distortion of the peptide from planarity. The distance between the active site serine O γ of SGPB and the carbonyl carbon of the scissile bond of PCI-1 is 2.8 Å (1 Å = 0.1 nm). The inhibitor has little secondary structure, having a three-stranded anti-parallel β-sheet on the side opposite the reactive site and four β-turns. PCI-1 has four disulphide bridges; these presumably take the place of extensive secondary structure in keeping the reactive site conformationally constrained. The pairing of the cystine residues, which had not been characterized chemically, is as follows: Cys3I to Cys40I, Cys6I to Cys24I, Cys7I to Cys36I, and Cys13I to Cys49I. The molecular structure of SGPB in the PCI-1 complex agrees closely with the structure of SGPB complexed with the third domain of the turkey ovomucoid inhibitor (OMTKY3). A least-squares overlap of all atoms in SGPB gives a root-mean-square difference of 0.37 Å. One of the loops of SGPB (Ser35 to Gly40) differs in conformation in the two complexes by more than 2.0 Å root-mean-square for the main-chain atoms. Thr39 displays the largest differences with the carbonyl carbon atom deviating by 3.6 Å. This conformational alternative is a result of the differences in the molecular structures of the P′ 4 residues following the reactive site bonds of the two inhibitors. This displacement avoids a close contact (1.3 Å) between the carbonyl oxygen of Ser38 of SGPB and Pro42I C β of PCI-1. The solvent structure of the PCI-1-SGPB complex includes 179 waters, two sulphate or phosphate ions, and one calcium or potassium ion, which appears to play a role in crystal formation. The molecular structure of PCI-1 determined here has allowed the proposal of a model for the structure of a two-domain inhibitor from potatoes and tomatoes, inhibitor II. The two domains of the model are related by a pseudo-2-fold axis between the three-stranded β-sheet of each domain.

Determination of the cleavage specificity of Streptomyces griseus protease B in the presence of guanidinium chloride.

Streptomyces griseus protease B (SGPB), a Pronase enzyme, has been shown to be stable and active in the presence of 6.0 M guanidinium chloride (Siegel, S. et al. (1972) J. Biol. Chem. 247, 4155-4159). In order to determine the cleavage specificity of this unusual enzyme under denaturing conditions, 12 peptides of known amino acid sequence were hydrolyzed by SGPB in the absence and presence of 6.0 M guanidinium chloride. The new N-terminal amino acids produced by the action of SGPB were dansylated and quantitatively identified by reverse phase HPLC. The results indicate that SGPB retained its cleavage specificity for phenylalanyl, tyrosyl, tryptophanyl, and leucyl peptide bonds in the presence of 6.0 M guanidinium chloride. Of these peptide bonds, SGPB exhibited a greater cleavage preference for phenylalanyl and tryptophanyl bonds, which was relatively unaffected by the presence of the denaturant. The SGPB-catalyzed cleavages of the leucyl peptide bonds examined (Leu-Met, Leu-Arg, Leu-Val, Leu-Thr, and Leu-Ile) were substantially decreased under denaturing conditions, while Leu-Gly bond cleavage by SGPB was virtually unaffected by denaturant. The demonstrated predictability of the catalytic preference of this unusual protease for phenylalanyl, tyrosyl, tryptophanyl, and leucyl-glycine peptide bonds under denaturing conditions enhances its utility in the site-specific proteolysis of insoluble or otherwise proteolysis-resistant polypeptide substrates.

Structure of the complex of Streptomyces griseus protease B and the third domain of the turkey ovomucoid inhibitor at 1.8-A resolution.

The structure of the complex between the serine protease Streptomyces griseus protease B (SGPB) and the third domain of the Kazal-type ovomucoid inhibitor from turkey has been solved at 1.8-A resolution and refined to a conventional R factor of 0.125. As others have reported previously for analogous complexes of proteases and protein inhibitors, the inhibitor binds in a fashion similar to that of a substrate; it is not cleaved, but there is a close approach (2.7 A) of the active site nucleophile Ser-195 O gamma to the carbonyl carbon of the reactive peptide bond of the inhibitor. Contrary to the structural reports regarding the other enzyme-inhibitor complexes, we conclude that there is no evidence for a significant distortion of this peptide bond from planarity. The mechanism of inhibition can be understood in terms of the equilibrium thermodynamic parameters Ka, the enzyme-inhibitor association constant, and Khyd, the equilibrium constant for inhibitor hydrolysis. These thermodynamic parameters can be rationalized in terms of the observed structure.

Refined crystal structure of the molecular complex of Streptomyces griseus protease B, a serine protease, with the third domain of the ovomucoid inhibitor from turkey.

We have determined the crystal structure of the molecular complex between Streptomyces griseus protease B (SGPB), a bacterial serine protease, and the third domain of the ovomucoid inhibitor from turkey. Restrained-parameter least-squares refinement of the structure with the 1.8-A intensity data set has resulted in an R factor of 0.125. The carbonyl carbon atom of the reactive bond between Leu-18 and Glu-19 in the inhibitor lies at a distance of 2.71 A from the O gamma atom of the nucleophilic Ser-195 in SGPB; this distance is 0.5 A shorter than a normal van der Waals contact. Unlike the reactive bond in the pancreatic trypsin inhibitor complexed with bovine trypsin, the Leu--Glu bond of the ovomucoid inhibitor is not distorted from planarity towards a pyramidal configuration.

The 2.8 A resolution structure of Streptomyces griseus protease B and its homology with alpha-chymotrypsin and Streptomyces griseus protease A.

The 2.8 A (1 A = 0.1 nm) resolution structure of the crystalline orthorhombic form of the microbial serine protease Streptomyces griseus protease B (SGPB) has been solved by the method of multiple isomorphous replacement using five heavy-atom derivatives. The geometrical arrangement of the active site quartet, Ser-214, Asp-102, His-57, and Ser-195, is similar to that found for pancreatic alpha-chymotrypsin. SGPB and alpha-chymotrypsin have only 18% identity of primary structure but their tertiary structures are 63% topologically equivalent within a root mean square deviation of 2.07 A. The major tertiary structural differences between the bacterial enzyme SGPB and the pancreatic enzymes is due to the zymogen requirement of the multicellular organisms in order to protect themselves against autolytic degradation. The two pronase enzymes, SGPB and Streptomyces griseus protease A (SGPA), have 61% identity of sequence and their tertiary structures are 85% topologically equivalent within a root mean square deviation of 1.46 A. The active site regions of SGPA and SGPB are similar and their tertiary structures differ only in three minor regions of surface loops.