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1.
A systematic series of synthetic chromophoric substrates for aspartic proteinases. 总被引:10,自引:1,他引:9
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B M Dunn M Jimenez B F Parten M J Valler C E Rolph J Kay 《The Biochemical journal》1986,237(3):899-906
The hydrolysis of the chromogenic peptide Pro-Thr-Glu-Phe-Phe(4-NO2)-Arg-Leu at the Phe-Phe(4-NO2) bond by nine aspartic proteinases of animal origin and seven enzymes from micro-organisms is described [Phe(4-NO2) is p-nitro-L-phenylalanine]. A further series of six peptides was synthesized in which the residue in the P3 position was systematically varied from hydrophobic to hydrophilic. The Phe-Phe(4-NO2) bond was established as the only peptide bond cleaved, and kinetic constants were obtained for the hydrolysis of these peptide substrates by a representative selection of aspartic proteinases of animal and microbial origin. The value of these water-soluble substrates for structure-function investigations is discussed. 相似文献
2.
A Miteva A Karshikov B Atanasov A A Zhdanov N S Andreeva 《Molekuliarnaia biologiia》1988,22(6):1456-1463
A semi-empirical approach has been used to estimate the intramolecular electrostatic interactions in pepsin and penicillopepsin. The pH-dependence of the free energy electrostatic term was calculated, and the pH-dependence of the domain interactions has been estimated. As it was shown, the contribution of electrostatic interactions is rather small for the stabilization of the native structure. At the same time the electrostatic repulsion between domains increases with the increase of pH. The later can be the cause of the alkaline denaturation of pepsin and domain mobility. 相似文献
3.
A new assay for cathepsin B1 and other thiol proteinases 总被引:27,自引:0,他引:27
A J Barrett 《Analytical biochemistry》1972,47(1):280-293
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5.
Benzyloxycarbonylphenylalanylcitrulline p-nitroanilide as a substrate for papain and other plant cysteine proteinases.
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After preliminary assays, with papain, bromelain and ficin, on a range of citrulline p-nitroanilides, values of Km and kcat. for the papain-catalysed hydrolysis of three derivatives, N alpha- benzyloxycarbonylcitrulline p-nitroanilide, benzyloxycarbonylphenylalanylcitrulline p-nitroanilide and benzyloxycarbonylglycylphenylalanylcitrulline p-nitroanilide, were obtained. It is concluded that benzyloxycarbonylphenylalanylcitrulline p-nitroanilide is a highly selective substrate for the sensitive detection and assay of the plant cysteine proteinases. 相似文献
6.
Two chimeric enzymes were constructed by exchanging domains between porcine pepsinogen and rhizopuspepsinogen in order to examine the contributions of the subsites present on different domains toward enzymatic specificity. Both chimeras exhibited the characteristic features of aspartic proteinases, such as auto-activation at low pH and abrogation of enzymatic activity by pepstatin. The activity of the chimera containing the N-terminal domain of rhizopuspepsinogen and the C-terminal domain of porcine pepsinogen (rhzNppC) could be observed by HPLC after prolonged incubation with the substrates. In contrast, the reciprocal chimera, ppNrhzC, containing the N-terminal domain of porcine pepsinogen and the C-terminal domain of rhizopuspepsinogen exhibited catalytic activity, measurable by a spectrophotometric assay. Kinetic data and inhibitor analyses strongly suggest that interdependency may exist between adjacent subsites contributed by different domains. Therefore, in order to develop an optimal substrate or inhibitor, the effect of adjacent residues of the ligand has to be examined along with the preferences for each subsite. 相似文献
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《Revista iberoamericana de micología》2016,33(2):76-82
The aspartic proteases, also called aspartyl and aspartate proteases or acid proteases (E.C.3.4.23), belong to the endopeptidase family and are characterized by the conserved sequence Asp-Gly-Thr at the active site. These enzymes are found in a wide variety of microorganisms in which they perform important functions related to nutrition and pathogenesis. In addition, their high activity and stability at acid pH make them attractive for industrial application in the food industry; specifically, they are used as milk-coagulating agents in cheese production or serve to improve the taste of some foods. This review presents an analysis of the characteristics and properties of secreted microbial aspartic proteases and their potential for commercial application. 相似文献
9.
Structure and function of plant aspartic proteinases. 总被引:6,自引:0,他引:6
Aspartic proteinases of the A1 family are widely distributed among plant species and have been purified from a variety of tissues. They are most active at acidic pH, are specifically inhibited by pepstatin A and contain two aspartic residues indispensible for catalytic activity. The three-dimensional structure of two plant aspartic proteinases has been determined, sharing significant structural similarity with other known structures of mammalian aspartic proteinases. With a few exceptions, the majority of plant aspartic proteinases identified so far are synthesized with a prepro-domain and subsequently converted to mature two-chain enzymes. A characteristic feature of the majority of plant aspartic proteinase precursors is the presence of an extra protein domain of about 100 amino acids known as the plant-specific insert, which is highly similar both in sequence and structure to saposin-like proteins. This insert is usually removed during processing and is absent from the mature form of the enzyme. Its functions are still unclear but a role in the vacuolar targeting of the precursors has been proposed. The biological role of plant aspartic proteinases is also not completely established. Nevertheless, their involvement in protein processing or degradation under different conditions and in different stages of plant development suggests some functional specialization. Based on the recent findings on the diversity of A1 family members in Arabidopsis thaliana, new questions concerning novel structure-function relationships among plant aspartic proteinases are now starting to be addressed. 相似文献
10.
Engineering the substrate specificity of rhizopuspepsin: the role of Asp 77 of fungal aspartic proteinases in facilitating the cleavage of oligopeptide substrates with lysine in P1.
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W. T. Lowther P. Majer B. M. Dunn 《Protein science : a publication of the Protein Society》1995,4(4):689-702
Rhizopuspepsin and other fungal aspartic proteinases are distinct from the mammalian enzymes in that they are able to cleave substrates with lysine in the P1 position. Sequence and structural comparisons suggest that two aspartic acid residues, Asp 30 and Asp 77 (pig pepsin numbering), may be responsible for generating this unique specificity. Asp 30 and Asp 77 were changed to the corresponding residues in porcine pepsin, Ile 30 and Thr 77, to create single and double mutants. The zymogen forms of the wild-type and mutant enzymes were overexpressed in Escherichia coli as inclusion bodies. Following solubilization, denaturation, refolding, activation, and purification to homogeneity, structural and kinetic comparisons were made. The mutant enzymes exhibited a high degree of structural similarity to the wild-type recombinant protein and a native isozyme. The catalytic activities of the recombinant proteins were analyzed with chromogenic substrates containing lysine in the P1, P2, or P3 positions. Mutation of Asp 77 resulted in a loss of 7 kcal mol-1 of transition-state stabilization energy in the hydrolysis of the substrate containing lysine in P1. An inhibitor containing the positively charged P1-lysine side chain inhibited only the enzymes containing Asp 77. Inhibition of the Asp 77 mutants of rhizopuspepsin and several mammalian enzymes was restored upon acetylation of the lysine side chain. These results suggest that an exploitation of the specific electrostatic interaction of Asp 77 in the active site of fungal enzymes may lead to the design of compounds that preferentially inhibit a variety of related Candida proteinases in immunocompromised patients. 相似文献
11.
A convenient chromophoric assay for porcine pepsin has been developed using a new synthetic substrate. The sequence of this substrate was chosen based on the known subsite preferences for this enzyme. The peptide contains a phenylalanyl-p-nitrophenylalanine sequence at the reactive site. Cleavage of this bond yields a change in absorbance at 310 nm of between 1700 and 2000 per mole. This allows kinetic data to be obtained readily and accurately. The products of cleavage have been identified by isolation of a peptide fragment by high-performance liquid chromatography. Values of kcat, Km, and kcat/Km of 94 +/- 6 s-1, 0.13 +/- .04 mM, and 815 +/- 210 s-1/mM-1 were obtained at pH 3.0 and 37 degrees C. The peptide is soluble over the pH range from 2 to 7, thus facilitating determination of the pH dependence of the kinetic parameters. The substrate is also valuable in studying the inhibition of pepsin. 相似文献
12.
Structural and evolutionary relationships between retroviral and eucaryotic aspartic proteinases. 总被引:9,自引:0,他引:9
Three-dimensional crystal structures of the homologous retroviral proteinases from Rous sarcoma virus (RSV PR) and from human immunodeficiency virus (HIV-1 PR) are to a large extent similar and bear close resemblance to the six known structures of the bilobal fungal and mammalian aspartic proteinases. Systematic three-dimensional structural superpositions were carried out between the retroviral and the eucaryotic aspartic proteinases. Both retroviral enzymes were found to be similarly related to their fungal and mammalian counterparts. The most strongly conserved parts correspond to those regions in the N- and C-domains of the eucaryotic enzymes that are related by the interdomain dyad and consist of a combination of secondary structural elements that form the psi loop-alpha helix motif at the active sites of the aspartic proteinases. The retroviral proteinase monomer exhibits nearly the same degree of structural equivalence to the N- and C-domains of the eucaryotic enzymes. In light of the deduced structural relationships between HIV-1 PR and RSV PR, sequence alignments were performed for a number of retroviral proteinases from different subfamilies. There are three highly conserved amino acid sequence stretches, of which two belong to the psi loop-alpha helix motif, that bear moderate sequence similarity with the eucaryotic enzymes. The third conserved sequence stretch among the retroviral proteinases belongs to the flap and bears no resemblance to the flap sequences in the eucaryotic enzymes. The interdomain antiparallel beta sheet in the cellular enzymes differs from the intersubunit beta sheet in the number, arrangement, and directionality of strands, suggesting the possibility of convergent evolution.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
13.
The amino acid sequence of Mucor pusillus aspartic proteinase was determined by analysis of fragments obtained from cleavage of the enzyme by CNBr and limited tryptic digestion. The proteinase is a single polypeptide chain protein containing 361 amino acid residues, cross-linked by two disulfide bonds. A sugar moiety composed of two GlcNAc residues and four neutral sugar residues is asparagine-linked to the chain. The sequence of M. pusillus proteinase is highly homologous with the M. miehei proteinase (83% identity). The homology with other aspartic proteinases is low (22-24%) and indicates that the Mucor proteinases diverged at an early evolutionary phase. The most conservative regions of the molecule are those involved in catalysis and forming the binding cleft and the core region of the molecule. 相似文献
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Kucerová Z Tichá M 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2002,770(1-2):121-128
Affinity chromatography was used to study an interaction of aspartic proteinases with immobilized aromatic amino acids and their derivatives. The following ligands were used: L-tyrosine, 3-iodo-L-tyrosine, 3,5-diiodo-L-tyrosine, L-phenylalanine, p-iodo-L-phenylalanine and N-acetyl-L-phenylalanine. With the exception of the last one, ligands were coupled directly to divinyl sulfone activated Sepharose 4B. For the preparation of immobilized N-acetyl-L-phenylalanine, divinyl sulfone activated Sepharose 4-B with linked ethylene diamine was used. Porcine pepsin was used for the evaluation of the capacity of the prepared affinity carriers. The capacity of the immobilized amino acid derivatives significantly increased in comparison with the non-derivatized amino acids. The prepared immobilized ligands were further used for the separation of human pepsinogens. 相似文献
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Allan Beveridge 《Proteins》1996,24(3):322-334
We have performed ab initio Hartree-Fock self-consistent field calculations on the active site of endothiapepsin. The active site was modeled as a formic acid/formate anion moiety (representing the catalytic aspartates, Asp-32 and -215) and a bound water molecule. Residues Gly-34, Ser-35, Gly-217, and Thr-218, which all form hydrogen bonds to the active site, were modeled using formamide and methanol molecules. The water molecule, which is generally believed to function as the attacking nucleophile in catalysis, was allowed to bind to the active site in four distinct configurations. The geometry of each configuration was optimized using two basis sets (4-31G and 4-31G*). The results indicate that in the native enzyme the nucleophilic water is bound in a catalytically inert configuration. However, by rotating the carboxyl group of Asp-32 by about 90° the water molecule can be reorientated to attack the scissile bond of the substrate. A model of the bound enzyme-substrate complex was constructed from the crystal structure of a difluorostatone inhibitor complexed with endothiapepsin. This model suggests that the substrate itself initiates the reorientation of the nucleophilic water immediately prior to catalysis by forcing the carboxyl group of Asp-32 to rotate. The theoretical results predict that the active site of endothiapepsin undergoes a large distortion during substrate binding and this observation has been used to explain some of the kinetics results which have been reported for mutant aspartic proteinases. 相似文献
18.
The mammalian aspartic proteinases procathepsin D and pepsinogen form insoluble inclusion bodies when expressed in bacteria. They become soluble but nonnative when synthesized as fusions to the carboxy terminus of E. coli maltose-binding protein (MBP). Since these nonnative states of the two aspartic proteinases showed no tendency to form insoluble aggregates, their biophysical properties were analyzed. The MBP portions were properly folded as shown by binding to amylose, but the aspartic proteinase moieties failed to bind pepstatin and lacked enzymatic activity, indicating that they were not correctly folded. When treated with proteinase K, only the MBP portion of the fusions was resistant to proteolysis. The fusion between MBP and cathepsin D had increased hydrophobic surface exposure compared to the two unfused partners, as determined by bis-ANS binding. Ultracentrifugal sedimentation analysis of MBP–procathepsin D and MBP–pepsinogen revealed species with very large and heterogeneous sedimentation values. Refolding of the fusions from 8 M urea generated proteins no larger than dimers. Refolded MBP–pepsinogen was proteolytically active, while only a few percent of renatured MBP–procathepsin D was obtained. The results suggest that MBP–aspartic proteinase fusions can provide a source of soluble but nonnative folding states of the mammalian polypeptides in the absence of aggregation. 相似文献
19.
Milisavljevic MDj Timotijevic GS Radovic SR Konstantinovic MM Maksimovic VR 《Journal of plant physiology》2008,165(9):983-990
Two types of aspartic proteinase (AP) genes have been isolated from the cDNA library of developing buckwheat seeds. Analysis of their sequences showed that one of these, FeAP9, resembled the structure and shared high homology with the so-called typical plant APs characterized by the presence of a plant-specific insert (PSI), an element unique among APs. The other cDNA, FeAPL1, encoded an AP-like protein lacking that domain. Different expression profiles were observed for FeAP9 and FeAPL1. FeAPL1 mRNAs were restricted to the seeds only, whereas FeAP9 mRNAs were also present in the other plant tissues - leaves, roots, and flowers. Higher levels of FeAP9 were observed in senescent leaves compared with green leaves. The differential expression pattern of these two unique APs raises the interesting possibility that these proteinases have unique substrate specificity and may have different roles in plant development and other physiological processes. 相似文献
20.
A new chromogenic substrate for subtilisin 总被引:2,自引:0,他引:2
L A Lyublinskaya S V Belyaev A Y Strongin L F Matyash E D Levin V M Stepanov 《Analytical biochemistry》1974,62(2):371-376
A new chromogenic substrate, benzyloxycarbonyl-glycyl-glycyl-l-leucine p-nitroanilide, was synthesiszed allowing convenient spectrophotometric assay of subtilisin activity. This substrate has been applied to the determination of subtilisin in solution as well as for the identification of subtilisin after gel electrophoresis or isoelectrophocusing. Catalytic parameters for the new substrate were determined. 相似文献