Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A homogeneous serine proteinase was isolated from cultural filtrates of the extreme halophilic bacteria Halobacterium mediterranei 1538 using affinity chromatography on bacitracin-Sepharose, ultrafiltration and gel filtration on Sephadex G-75, with a 48% yield and 260-fold purification. The enzyme was completely inactivated by specific inhibitors of serine proteinases, PMSF and DFP, as well as by Hg2+ and PCMB. The enzyme activity was strongly dependent of NaCl concentration, the enzyme being inactivated below 0.75 M NaCl. Inactivation of the enzyme was also seen in the presence of 2-7% organic solvents. The pH optimum for Glp-Ala-Ala-Leu-pNA hydrolysis is 8.0-8.5; Km is 0.14 mM, kcat is 36.9 s-1. The stability optimum lies at pH 5.5-8.0, temperature optimum is at 55 degrees C. The enzyme molecular weight is 41,000 Da; pI is 7.5. The substrate specificity of the enzyme is comparable to that of secretory subtilisins; the extent of protein substrate hydrolysis is similar to that of proteinase K. The N-terminal sequence of Halobacterium mediterranei serine proteinase, Asp-Thr-Ala-Asn-Asp-Pro-Lys-Tyr-Gly-Ser-Gln-Tyr-Ala-Pro-Gln-Lys-Val-Asn- Ala- Asp-, reveals a 50% homology with the aminoterminal sequence of Thermoactinomyces vulgaris serine proteinase. Hence, the serine proteinase secreted by halophilic bacteria may be considered as a structural and functional analog of eubacterial enzymes.
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PMID:[Serine proteinase from the archaebacterium Halobacterium mediterranei--an analog of eubacterium subtilisin]. 139 Dec 25

Heparan sulfate binds to proteins present on the surface of Staphylococcus aureus cells. Binding of 125I-heparan sulfate to S. aureus was time dependent, saturable, and influenced by pH and ionic strength, and cell-bound 125I-heparan sulfate was displaced by unlabelled heparan sulfate or heparin. Other glycosaminoglycans of comparable size (chondroitin sulfate and dermatan sulfate), highly glycosylated glycoprotein (hog gastric mucin), and some anionic polysaccharides (dextran sulfate and RNA) inhibited heparan sulfate binding to various extents. Heat treatment (80 degrees C for 10 min) and treatment of the bacteria with pronase E, proteinase K, pepsin, and chymotrypsin considerably reduced their ability to bind 125I-heparan sulfate, but treatment with trypsin and neuraminidase did not affect binding. Scatchard plot analysis indicated the presence of cell surface components with low affinity (Kd = 3 x 10(-5) M) for heparan sulfate. Cell surface components were released by stirring bacteria with 1 M LiCl at 37 degrees C for 2 h. Proteins of this extract that competitively inhibited binding of 125I-heparan sulfate to S. aureus were isolated by affinity chromatography on heparin-Sepharose. Two proteins having molecular masses of approximately 66 and 60 kDa and the ability to bind 125I-heparan sulfate were obtained. The first 9 amino-terminal amino acid residues of the 66-kDa protein are Asp-Trp-Thr-Gly-Trp-Leu-Ala-Ala-Ala, and the first 4 amino-terminal amino acid residues of the 60-kDa protein are Met-Leu-Val-Thr.
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PMID:Binding of heparan sulfate to Staphylococcus aureus. 154 63

The three-dimensional crystal structure of thermitase complexed with eglin-c in the presence of 100 mM calcium has been determined and refined at 2.0-A resolution to a R-factor of 16.8%. This crystal structure is compared with previously determined structures of thermitase at 0 and 5 mM calcium concentration. In the presence of 100 mM calcium all three calcium binding sites in thermitase are fully occupied. At 100 mM CaCl2 the "weak" calcium binding is occupied by a calcium ion, which is chelated by three protein ligands and four water molecules in a pentagonal bipyramid geometry. Thermitase has, apparently, a monovalent and divalent cation binding position at 2.5-A distance from each other at this site. At low calcium concentrations the monovalent-ion position is occupied by a sodium or potassium ion. The "medium strength" binding site shows in the presence of 100 mM CaCl2 a square antiprism arrangement with eight ligands, of which seven are donated by the protein. At low calcium concentrations we observe a distorted pentagonal bipyramid coordination at this site. The largest difference between these two conformations is observed for ligand Asp-60, which has two conformations with 0.8-A difference in C alpha positions. The "strong" calcium binding site has a pentagonal bipyramid coordination and is fully occupied in all three structures. Structural changes on binding calcium to the weak and "medium strength" calcium binding sites of thermitase are limited to the direct surroundings of these sites. Thermitase resembles in this respect subtilisin BPN' and does not exhibit long-range shifts as have been reported for proteinase K.
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PMID:Calcium binding to thermitase. Crystallographic studies of thermitase at 0, 5, and 100 mM calcium. 199 69

The X-ray crystal structure of the subtilisin-type enzyme proteinase K at 1.5 A resolution shows that is has two binding sites for Ca2+. Scatchard analysis indicates that one Ca2+ binds tightly, with pK 7.6 x 10(-8) M-1, and the other only weakly. Although Ca2+ is not directly involved in the catalytic mechanism and is 16.6 A away from the alpha-carbon atoms of the catalytic triad Asp 39-His 69-Ser 224, the activity of proteinase K towards the synthetic substrate succinyl-Ala-Ala-Ala-p-nitroanilide drops slowly to approximately 20% of its original value when it is depleted of Ca2+. This is not due to autolysis of the enzyme. The X-ray crystal structure of Ca2+-free proteinase K shows that removal of Ca2+ from the tight binding site triggers a concerted domino-like movement of five peripheral loops and of two alpha-helices. At a distance of 25 A from this calcium-binding site, the geometry of both the secondary substrate binding site and of the catalytic triad is affected by this movement thereby reducing the activity of the enzyme.
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PMID:Long-range structural changes in proteinase K triggered by calcium ion removal. 291 93

Proteolysis of glucose dehydrogenase from Bacillus megaterium with proteinase K apparently generated two fragments. The small fragment, designated as K-peptide, was sequenced and its covalent structure was determined as Ser-Ser-Glu-Ala-Ser-Tyr-Val-Thr-Gly-Ile-Thr-Leu-Phe-Ala-Asp-Gly-Gly-Met-Thr- Gln-Tyr-Pro-Ser-Phe-Glu-Ala-Gly-Arg-Gly. The sequence analysis showed that the K-peptide consists of two identical fragments, one of which is lacking one serine residue at the amino-terminus.
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PMID:Amino acid sequence of the K-peptide generated by limited proteolysis of glucose dehydrogenase from Bacillus megaterium by proteinase K1. 642 50

The hydrolytic cleavage of a cyanine (Cy3)-labeled angiotensin, catalyzed by various proteases, was studied by capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF). The end-labeled peptides and the Cy3 diacid internal standard were separated on a 20-microns x 27-cm capillary with LIF detection (emission, 580 nm) using a frequency-doubled solid-state diode laser emitting at 532 nm or a He-Ne laser emitting at 543 nm. Hydrolysis of the Cy3-labeled angiotensin I, catalyzed by proteinase K, is a sequential process beginning from the C-terminal of the peptide, instead of from random cleavages. Trypsin catalyzes a specific cleavage of Cy3-angiotensin I to Cy3-Asp-Arg as anticipated. Using a combination of endopeptidase and carboxypeptidases, the remnant of the labeled species was characterized by CE-LIF. The method provides a general tool for studying the mechanism of protease-catalyzed hydrolysis of peptide.
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PMID:Characterization of protease-catalyzed hydrolysis of cyanine-labeled angiotensin using capillary electrophoresis with laser-induced fluorescence detection. 776 1

Fatal familial insomnia (FFI) and a subtype of familial Creutzfeldt-Jakob disease (CJD178) are two prion diseases that have different clinical and pathological features, the same aspartic acid to asparagine mutation (D178N) at codon 178 of the prion protein (PrP) gene, but distinct genotypes generated by the methionine-valine polymorphism at codon 129 (129M or 129V) in the mutant allele of the PrP gene. The D178N, 129M allele segregates with FFI while the D178N, 129V allele segregates with CJD178. The proteinase K resistant PrP (PrPres) isoforms present in FFI and CJD178 differ in degree of glycosylation and size. Thus, the amino acid, methionine or valine, at position 129 of the mutant allele, in conjunction with D178N mutation results in significant alterations of PrPres in FFI and CJD178. The 129 polymorphic site also exerts influence through the normal allele: the course of the disease is shorter in the patients homozygous at codon 129 and other minor but consistent phenotypic differences occur between homozygous and heterozygous FFI patients. The comparative study of PrPres distribution in FFI homozygotes and heterozygotes at codon 129 has lead to the conclusion that the phenotypic differences observed between these two FFI patient populations may be the result of different rates of conversion of normal PrP into PrPres, at least in some brain regions.
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PMID:Fatal familial insomnia and familial Creutzfeldt-Jakob disease: clinical, pathological and molecular features. 776 90

The melanin operon (melC) of Streptomyces antibioticus contains two genes, melC1 and melC2 (apotyrosinase). Our previous studies indicated that MelC1 forms a transient binary complex with the downstream apotyrosinase MelC2 to facilitate the incorporation of copper ion and the secretion of tyrosinase. In this study, we investigated the role of histidine residues in the function of MelC1 by examining a series of substitution or deletion mutants. Of eight mutants only the substitution of His-117 with Asp in the mutant M-117D rendered the complete abolishment of the intracellular tyrosinase activity in both Streptomyces and Escherichia coli. Replacement of His-102 by Leu in the mutant M-102L also caused a 64-70% reduction of tyrosinase activity in Streptomyces and E. coli. These two mutations also affected the secretion of both MelC1 and MelC2 proteins. In vitro copper activation of the purified MelC1.MelC2 binary complex from these two mutants regained only 20-30% tyrosinase activity of the wild type. Biochemical characterization of the tyrosinases from these two mutants revealed that they were different in several aspects. The intracellular tyrosinase activity in M-117D, but not in M-102L, could be partially reactivated by copper ion or by the cell extract containing MelC1. The copper content and the specific activity of the tyrosinase purified from the culture supernatant from M-117D were only 40% of those in wild type and M-102L. Additionally, fast protein liquid chromatography analysis indicated that in these two mutants the copper activation process was defective, very likely due to the incompetent MelC1.MelC2 binary complex formed: reduced association in M-117D and elevated association in M-102L. Furthermore, the conformation of MelC2 in the binary complex or in the mature enzyme form in wild type could be differentiated by the proteinase K digestion pattern, and so did the conformation of MelC2 found in those of M-102L, but not in M-117D mutant. Taken together, our results demonstrate that MelC1 is indispensable in the incorporation of copper ion into MelC2 apotyrosinase via a transient, competent binary complex formation, during which a conformational transition of MelC2 has occurred. This strongly suggests that MelC1 is a chaperone for the apotyrosinase MelC2.
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PMID:Mutational study of Streptomyces tyrosinase trans-activator MelC1. MelC1 is likely a chaperone for apotyrosinase. 836 Jan 64

Hot water extract prepared from the mycelial culture of mushroom Phellinus linteus stimulated polyclonal antibody production in an in vitro culture system. The active fraction PLP was purified from the extract ca. 1030-fold by ethanol precipitation followed by DEAE-cellulose and gel permeation chromatography. PLP contained 13.2% (w/w) peptide and 82.5% (w/w) carbohydrate. About 6.8% (w/w) of the total carbohydrate was uronic acid. The molecular weight distribution of PLP was found to be nearly homogeneous (153 kDa) in gel permeation HPLC analysis. Neutral sugar composition analysis revealed Ara (7.5%), Xyl (3.7%), Glc (21.1%), Gal (24.1%) and Man (44.2%). Uronic acid was identified as a glucuronic acid by gas chromatography. Ten amino acids were detected and Asp and Glu were the major components. In our assay system, the half-maximal concentration of PLP for B-lymphocyte stimulation was ca. 3 micrograms/ml. Partial acid hydrolysis as well as sodium periodate treatment of PLP decreased the activity significantly, suggesting that both the full molecular size and the sugar moiety were essential. However, proteinase K treatment for up to 48 h did not affect the activity.
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PMID:B-lymphocyte-stimulating polysaccharide from mushroom Phellinus linteus. 858 12

The crystal structure of a ternary complex of proteinase K, Hg(II) and a hexapeptide N-Ac-Pro-Ala-Pro-Phe-Pro-Ala-NH2 has been determined at 2.2 A resolution and refined to an R factor of 0.172 for 12,910 reflections. The mercury atom occupies two alternate sites, each of which was assigned an occupancy of 0.45. These two sites are bridged by Cys-73 S gamma which forms covalent bonds to both. Both mercury sites form regular polyhedrons involving atoms from residues Asp-39, His-69, Cys-73, His-72, Met-225, and Wat-324. The complex formation with mercury seems to disturb the stereochemistry of the residues of the catalytic triad Asp-39, His-69, and Ser-224 appreciably, thus reducing the enzymatic activity of proteinase K to 15%. The electron density in the difference Fourier map shows that the hexapeptide occupies the S1 subsite predominantly and the standard recognition site constituted by Ser-132 to Gly-136 and Gly-100 to Tyr-104 segments is virtually empty. The hexapeptide is held firmly through a series of hydrogen bonds involving protein atoms and water molecules. As a result of complex formation, Asp-39, His-69, Met-225, Ile-220, Ser-219, Thr-223, and Ser-224 residues move appreciably to accommodate the mercury atoms and the hexapeptide. The largest movement is observed for Met-225 which is involved in multiple interactions with both mercury and the hexapeptide. The activity results indicate an inhibition rate of 95%, as a result of the combined effect of mercury and hexapeptide.
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PMID:Structure of a ternary complex of proteinase K, mercury, and a substrate-analogue hexa-peptide at 2.2 A resolution. 881 35


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