| 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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MedLine Citation:
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PMID: 7613467 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
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Authors:
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W T Lowther; P Majer; B M Dunn |
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Publication Detail:
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Type: Journal Article; Research Support, U.S. Gov't, P.H.S. |
Journal Detail:
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Title: Protein science : a publication of the Protein Society Volume: 4 ISSN: 0961-8368 ISO Abbreviation: Protein Sci. Publication Date: 1995 Apr |
Date Detail:
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Created Date: 1995-08-24 Completed Date: 1995-08-24 Revised Date: 2009-11-19 |
Medline Journal Info:
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Nlm Unique ID: 9211750 Medline TA: Protein Sci Country: UNITED STATES |
Other Details:
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Languages: eng Pagination: 689-702 Citation Subset: IM |
Affiliation:
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Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville 32610-0245, USA. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acid Sequence Animals Aspartic Acid / chemistry* Aspartic Acid Endopeptidases / antagonists & inhibitors, chemistry, genetics, metabolism* Base Sequence Circular Dichroism Computer Graphics Enzyme Inhibitors / pharmacology Enzyme Precursors / metabolism Hydrogen Bonding Hydrogen-Ion Concentration Kinetics Models, Molecular Molecular Sequence Data Mutagenesis, Site-Directed Oligopeptides / metabolism* Pepsin A / chemistry Protein Denaturation Protein Engineering* Protein Folding Recombinant Proteins / chemistry, metabolism Substrate Specificity Swine |
| Grant Support | |
ID/Acronym/Agency:
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DK18865/DK/NIDDK NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Enzyme Inhibitors; 0/Enzyme Precursors; 0/Oligopeptides; 0/Recombinant Proteins; 56-84-8/Aspartic Acid; EC 3.4.23.-/Aspartic Acid Endopeptidases; EC 3.4.23.1/Pepsin A; EC 3.4.23.21/rhizopuspepsin |
| Comments/Corrections | |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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