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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 P 1
Author(s) -
Todd Lowther W.,
Dunn Ben M.,
Majer Pavel
Publication year - 1995
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560040409
Subject(s) - lysine , biochemistry , enzyme , zymogen , chemistry , acetylation , mutant , aspartic acid , escherichia coli , amino acid , stereochemistry , gene
Rhizopuspepsin and other fungal aspartic proteinases are distinct from the mammalian enzymes in that they are able to cleave substrates with lysine in the P 1 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 P 1 , P 2 , or P 3 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 P 1 . An inhibitor containing the positively charged P 1 ‐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.