NDLI: 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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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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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.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Majer, P. Dunn, B. M. Lowther, W. T.
Description	Country affiliation: United States Author Affiliation: Lowther WT ( Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville 32610-0245, USA.)
Abstract	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.
ISSN	09618368
e-ISSN	1469896X
Journal	Protein Science
Issue Number	4
Volume Number	4
Language	English
Publisher	Wiley-Blackwell (on behalf of The Protein Society)
Publisher Date	1995-04-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Aspartic Acid Endopeptidases Metabolism Aspartic Acid Chemistry Oligopeptides Protein Engineering Amino Acid Sequence Animals Antagonists & Inhibitors Genetics Circular Dichroism Computer Graphics Enzyme Inhibitors Pharmacology Enzyme Precursors Hydrogen Bonding Hydrogen-ion Concentration Kinetics Models, Molecular Molecular Sequence Data Mutagenesis, Site-directed Pepsin A Protein Denaturation Protein Folding Recombinant Proteins Substrate Specificity Swine Research Support, U.s. Gov't, P.h.s. Discipline Biochemistry
Content Type	Text
Resource Type	Article
Subject	Medicine Molecular Biology Biochemistry

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