Streptomyces Griseus Protease Research Articles

Arg15-Lys17-Arg18 turkey ovomucoid third domain inhibits human furin

Turkey ovomucoid third domain with Leu18 in its reactive site is a potent inhibitor of many serine proteinases: subtilisins, chymotrypsins, and elastases. Previous studies showed that an L18K mutation made it a moderately strong inhibitor of trypsin, while an L18E mutation made it a strong inhibitor of Glu-specific Streptomyces griseus proteinase (GluSGP). For human furin substrates the consensus optimal sequence is RXKR decreases. Therefore the A15R, T17K, and L18R mutations were made in turkey ovomucoid third domain. The mutant inhibits human furin with a Ka of 1.1 x 10(7) M-1. As human furin catalyzes an obligatory step in human immunodeficiency virus proliferation, this inhibitor, along with the others already available, deserves further study.

An accurate simulation of the protein Streptomyces griseus protease A

An accurate simulation of the protein Streptomyces griseus protease A

Synthetic analogues of chymostatin. Inhibition of chymotrypsin and Streptomyces griseus proteinase A.

A series of analogues of chymostatin, including Z-Arg-Leu-Phe-aldehyde (Z-Arg-Leu-Phe-H), have been synthesized. Analysis of the inhibitory potential of these analogues permits identification of residues and interactions that are important for inhibitory activity. Moreover, the structure-function relationship for Z-Arg-Leu-Phe-H and chymostatin inhibition of chymotrypsin and Streptomyces griseus proteinase A (SGPA) was probed further with the aid of molecular mechanics. This analysis identified interactions that provide an explanation for the enhanced activity of the natural product, chymostatin, over the synthetic analogues in the inhibition of chymotrypsin but not SGPA.

Protease pro region required for folding is a potent inhibitor of the mature enzyme.

alpha-Lytic protease, an extracellular bacterial serine protease, is synthesized with a large pro region that is required in vivo for the proper folding of the protease domain. To allow detailed mechanistic study, we have reconstituted pro region-dependent folding in vitro. The pro region promotes folding of the protease domain in the absence of other protein factors or exogenous energy sources. Surprisingly, we find that the pro region is a high affinity inhibitor of the mature protease. The pro region also inhibits the closely related Streptomyces griseus protease B, but not the more distantly related, yet structurally similar protease, elastase. Based on these data, we suggest a mechanism in which pro region binding reduces the free energy of a late folding transition state having native-like structure.

Effects of Organic Solvents on Streptomyces griseus Protease I with Transfer Action.

The protease I from Streptomyces griseus var. alcalophilus strongly catalyzed the transfer reaction forming hydroxamic acids of various amino acids, especially aromatic and hydrophobic amino acids. Dimethyl sulfoxide (DMSO), 1,4-butane diol, and glycerol protected this enzyme from heat denaturation. Moreover, only DMSO strongly activated the transfer action of protease I and the velocity of reaction was accelerated three times in the presence of 50% DMSO. The yield of transfer product strongly depended on reaction time and the concentrations of enzyme and substrates in the presence of DMSO. The velocity of the transfer reaction was faster than that of the hydrolysis reaction under the conditions of 0.1 M Phe-OEt, 0.75 M NH2OH, and 50% DMSO.

Characterization of Rarobacter faecitabidus Protease I, a Yeast-Lytic Serine Protease Having Mannose-Binding Activity

Rarobacter faecitabidus protease I, a yeast-lytic serine protease, was characterized in order to elucidate the mechanism of lysis of yeast cells by this enzyme. The N-terminal amino acid sequence of the enzyme was found to be homologous to those of Lysobacter enzymogenes alpha-lytic protease and Streptomyces griseus proteases A and B around the catalytic His residue, showing that it is a mammalian type serine protease. In a study of its substrate specificity, it preferentially hydrolyzed the ester of alanine among amino acid p-nitrophenylesters. It also efficiently hydrolyzed succinyl Ala-Pro-Ala p-nitroanilide, the specific synthetic substrate for pancreatic elastase. With oxidized insulin B-chain, it hydrolyzed almost exclusively the peptide bond between valine 18 and cysteic acid 19 in the early step of the reaction, and thereafter it partially hydrolyzed Val12-Glu13, Ala14-Leu15, and Leu15-Tyr16. These results indicate that Rarobacter protease I is elastase-like in its substrate specificity, preferentially hydrolyzing the peptide bond of aliphatic amino acids. Its affinity for yeast cells was also investigated, and while Rarobacter protease I was adsorbed by yeast cells, pancreatic elastase was not. This difference was thought to account for the failure of pancreatic elastase to lyse yeast cells, even though its specificity is similar to that of the yeast-lytic enzyme. Rarobacter protease I was adsorbed by a mannose-agarose column and specifically eluted from the column with a buffer containing D-mannose or D-glucose. These monosaccharides also inhibited its yeast-lytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Replacement of P1 Leu18 by Glu18 in the reactive site of turkey ovomucoid third domain converts it into a strong inhibitor of Glu-specific Streptomyces griseus proteinase (GluSGP)

Turkey ovomucoid third domain with P1 Leu18 at its reactive site is an excellent inhibitor of chymotrypsin and elastase and of many other serine proteinases with related specificities. Semisynthetic replacement of P1 Leu18 by Lys18 causes the expected change into a trypsin inhibitor. Strikingly, semisynthetic replacement P1 Leu18 to Glu18 changes turkey ovomucoid third domain into a powerful inhibitor of Glu-specific Streptomyces griseus proteinase, GluSGP. Of the 131 natural avian ovomucoid third domains we have sequenced none have P1 Glu18, but several avian ovomucoid first domains have P1 Glu24. They are weak to moderate inhibitors of GluSGP.

Heparin binds normally to platelets digested with streptomyces griseus protease

Heparin binds normally to platelets digested with streptomyces griseus protease

Two new extracellular serine proteases from Streptomyces fradiae

1. 1. Two new extracellular serine proteases have been purified to homogeneity from Streptomyces fradiae. 2. 2. On amino acid sequencing, striking homology is observed between the first enzyme and Streptomyces griseus Protease A, and the second enzyme and S. griseus trypsin. 3. 3. The sequence information shows for the first time that structurally and enzymatically related serine proteases are extracellularly expressed by different Streptomycetes. 4. 4. Differential keratinolytic substrate specificity among these two microbes are probably due to a difference in disulfide reduction capacity.

Molecular dynamics study of the structure and dynamics of a protein molecule in a crystalline ionic environment, Streptomyces griseus protease A

A large-scale molecular dynamics simulation of the behavior of a serine protease (Streptomyces griseus protease A) in a crystalline environment has been performed. All atoms (including hydrogens) of two protein molecules and the surrounding solvent of crystallization, consisting of both water and salt ions, were explicitly represented, and a relatively long range of interactions (up to 15 A) were included. The simulation is the longest so far reported for a protein in such an environment (60 ps). The use of the full crystalline environment allows a direct comparison of the structure and dynamic properties of the protein and surrounding solvent to be made with the experimental X-ray structure. Here we report the comparison of the protein structures and analyze the energetics of the system, including interaction with the aqueous environment. Subsequent papers will deal with other aspects of the simulation. The overall root mean square differences between the time-averaged molecular dynamics structure and that from crystallography, for all well-ordered, non-hydrogen atoms, are 1.67 and 1.25 A for the two molecules taken as the asymmetric unit. An extensive analysis of the conformation of substructural elements and individual residues and their deviation from experiment has revealed a strong influence of the ionic medium on their behavior. Implications of the results for free energy calculations and for future directions are also discussed.

Fluorescence-labeled Streptomyces subtilisin inhibitor: analysis of the interaction with subtilisin and Streptomyces griseus proteases

Specific labeling of the Lys-89 residue of Streptomyces subtilisin inhibitor (SSI) was carried out by fluorescein isothiocyanate. The dimeric structure of SSI, which is composed of identical subunits, was not changed upon modification and the resulting fluorescent SSI was fully active as an inhibitor. Induced conformational change in the vicinity of the modified residue of SSI followed by complexation with subtilisin was evidenced by fluorescence spectra. Dissociation of the subunits was also shown to be associated with the change in the fluorescence spectra.

Unusual fucoidin-binding properties of chymotrypsinogen and trypsinogen

Previous work (Jones, R. (1987) Cell Biol. Int. Reports 11, 833 and Jones et al. (1988) Development 102, 781–792) has shown that sperm proacrosin (the enzyme form of the acrosomal proteinase acrosin, EC 3.4.21.10) has the capacity to recognize and bind sulphated polysaccharides and that this property is important for the initial stages of fertilization in mammals. To investigate whether this behaviour is specific to proacrosin, a variety of other proteinases (chymotrypsinogen, trypsinogen, thrombin, elastase, plasminogen, pepsin, Streptomyces griseus proteinase and V8 proteinase from Staphylococcus aureus) were immobilized on nitrocellulose and probed with [125I]fucoidin. Only chymotrypsinogen and trypsinogen retained significant amounts of the probe with Kd values of 1.4 · 10−6 M and 3.0 · 10−5 M, respectively. Proteinase inhibitors were ineffective as blocking agents suggesting that enzymic activity is not involved in recognition. However, the tertiary structure of the proteins is important, since cleavage of intramolecular disulphide bonds with 2-mercaptoethanol reduced binding by 50–60%. Competition experiments with a variety of mono- and polysaccharides suggest that the number and disposition of sulphate groups is critical for interaction with basic residues on the protein. It is concluded that, like proacrosin, chymotrypsinogen and trypsinogen are bifunctional proteins.

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).

Electrostatic complementarity in molecular associations

In this paper, I attempt to summarize the main qualitative features of electrostatic complementarity and similarity, important determinants of molecular recognition. The two aspects, Coulombic and hydrophobic matching, can be formulated in terms of molecular electrostatic potentials and fields. The Coulombic aspect is equivalent to the requirement to produce a potential pattern in the host cavity that is opposite in sign to that emerging from a guest. Hydrophobic complementarity is best described by the similis simili gaudet principle. This means that field patterns near the interacting molecular surfaces must be of similar magnitude. The above rules, which may find useful application in molecular graphics, were studied for different cases of enzyme-ligand interactions in trypsin. A further example, a noncovalent structural model of the catalytic diad in Streptomyces Griseus protease A, supports the observation that the same molecular entities form similar associations even in different environments, as is the case in the complex of small species in a crystal and amino acid residues with structural water molecules in a protein.

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.

PREPARATION OF PROTEASE FROM MIXED RUMEN MICROORGANISMS AND ITS USE FOR THE IN VITRO DETERMINATION OF THE DEGRADABILITY OF TRUE PROTEIN IN FEEDSTUFFS

Mixed rumen microorganisms present in bovine rumen fluid were extracted with butanol-acetone to provide a dry powder which retained 75–80% of the proteolytic activity of strained rumen fluid (SRF). Sixty percent of the proteolytic activity of the powder was extracted with water and concentrated on an Amicon XM-300 filter to give a protease preparation which had about 30% of the activity present in the SRF. The protease preparation was used for the determination of the rates of feed protein degradation in vitro by incubating at pH 6.8 in 0.1 M potassium phosphate buffer and measuring the rate of amino acid (and ammonia) production by the ninhydrin method. The relative degradation rates of the true proteins from feedstuffs tested were: soybean meal > fish meal > linseed meal and blood meal > canola meal > corn gluten meal. Substituting Streptomyces griseus protease for the rumen protease gave results which were very different from those obtained with the rumen enzyme. The advantages of using the rumen protease over other in vitro methods and the practical applicability of the procedure are discussed. Key words: Rumen, protease, preparation, in vitro, feed protein degradability

Electrostatic complementarity in molecular aggregates: Part 6. The carboxylate-imidazolium diad in Streptomyces Griseus protease A and in the 1,1′-binaphthyl-2,2′-dicarboxylic acid dihydrate- imidazole adduct

The electrostatic environment of the carboxylate-imidazolium diad in the active site of Streptomyces Griseus proteinase A is compared with that in a mixed crystal composed of 1,1′-binaphthyl-2,2′-dicarboxylic acid, imidazole and water. While in a previous study the geometric analogy has been stressed, it is shown in this paper that the electrostatic potential and field of the surroundings of the diad are very similar in both systems. This feature allows the crystal to be used as a simple model, relatively easily amenable to experiment, for the interpretation of various physicochemical, especially nuclear magnetic resonance spectroscopic, properties detected for serine proteases in solution.

A survey of the effects of proteases and glycosidases on culture of rabbit morulae to blastocysts.

The effects of a range of commercially available proteases and glycosidases on blastocyst development and hatching were examined on rabbit embryos cultured from the morula stage in a defined medium supplemented with charcoal-treated bovine serum albumin. The proteases tested were trypsin, alpha-chymotrypsin, thrombin, elastase, plasmin, papain, clostripain, collagenase, Streptomyces griseus protease and cathepsin C. The glycosidases tested were neuraminidase, alpha-mannosidase, beta-galactosidase and hyaluronidase. None of these enzymes appeared to stimulate blastocyst growth. The only enzymes which digested the embryonic investments, the zona and mucin coat, sufficiently to cause complete blastocyst hatching were trypsin and Streptomyces griseus protease at relatively low concentrations (250 ng/ml) and chymotrypsin and elastase at higher concentrations.

Refined structure of α-lytic protease at 1.7 Å resolution analysis of hydrogen bonding and solvent structure

The structure of α-lytic protease, a serine protease produced by the bacterium Lysobacter enzymogenes, has been refined at 1.7 Å resolution. The conventional R-factor is 0.131 for the 14,996 reflections between 8 and 1.7 Å resolution with I ≥ 2 σ( I). The model consists of 1391 protein atoms, two sulfate ions and 156 water molecules. The overall root-meansquare error is estimated to be about 0.14 Å. The refined structure was compared with homologous enzymes α-chymotrypsin and Streptomyces griseus protease A and B. A new sequence numbering was derived based on the alignment of these structures. The comparison showed that the greatest structural homology is around the active site residues Asp102, His57 and Ser195, and that basic folding pathways are maintained despite chemical changes in the hydrophobic cores. The hydrogen bonds in the structure were tabulated and the distances and angles of interaction are similar to those found in small molecules. The analysis also revealed the presence of close intraresidue interactions. There are only a few direct intermolecular hydrogen bonds. Most intermolecular interactions involve bridging solvent molecules. The structural importance of hydrogen bonds involving the side-chain of Asx residues is discussed. All the negatively charged groups have a counterion nearby, while the excess positively charged groups are exposed to the solvent. One of the sulfate ions is located near the active site, whereas the other is close to the N terminus. Of the 156 water molecules, only seven are not involved in a hydrogen bond. Six of these have polar groups nearby, while the remaining one is in very weak density. There are nine internal water molecules, consisting of two monomers, two dimers and one trimer. No significant second shell of solvent is observed.

The 1.8 Å structure of the complex between chymostatin and Streptomyces griseus protease A : A model for serine protease catalytic tetrahedral intermediates

The naturally occurring serine protease inhibitor, chymostatin, forms a hemiacetal adduct with the catalytic Ser195 residue of Streptomyces griseus protease A. Restrained parameter least-squares refinement of this complex to 1.8 Å resolution has produced an R index of 0.123 for the 11,755 observed reflections. The refined distance of the carbonyl carbon atom of the aldehyde to O γ of Ser195 is 1.62 Å. Both the R and S configurations of the hemiacetal occur in equal populations, with the end result resembling the expected configuration for a covalent tetrahedral product intermediate of a true substrate. This study strengthens the concept that serine proteases stabilize a covalent, tetrahedrally co-ordinated species and elaborates those features of the enzyme responsible for this effect. We propose that a major driving force for the hydrolysis of peptide bonds by serine proteases is the non-planar distortion of the scissile bond by the enzyme, which thereby lowers the activation energy barrier to hydrolysis by eliminating the resonance stabilization energy of the peptide bond.