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

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.

X‐ray structures of Sap1 and Sap5: Structural comparison of the secreted aspartic proteinases from Candida albicans

“…Aspartic proteinases in general prefer substrates with hydrophobic residues at positions P1 and P1′ for cleavage 48. However, fungal aspartic proteinases differ from mammalian enzymes in that they also cleave substrates with polar residues such as histidine or lysine in the P1 position 49…”

Recombinant immobilized rhizopuspepsin as a new tool for protein digestion in hydrogen/deuterium exchange mass spectrometry

“…Expression of Rpg from E. coli BL21 (DE3) (Novagen) cells harbouring the plasmid pET3a‐ rpg was realized as described by Pelosi et al 19 Protein purification from inclusion bodies was performed according to the protocol of Lowther et al ,20 modified by Flentke et al 21 The inclusion bodies were refolded by using the protocol of Flentke et al ,21 modified as follows. The refolded Rpg solution was centrifuged at 24 000 g for 30 min to remove insoluble material and aliquots of the refolded Rpg (1.7 mg/mL) were stored without Triton X‐100 at −20°C.…”

Study of Inhibitors Against SARS Coronavirus by Computational Approaches

“…The isostere has been incorporated into the ras farnesyl-protein transferase inhibitor that is a potential anticancer drug (Graham et al, 1994). Pseudo peptide analogs containing reduced amide isostere are also shown to inhibit C α -N-C cleavage in the gag region of HIV-1 protease (Lowther et al, 1995). It was also observed by quantum chemistry calcula-tions ) that after CO was replaced by CH 2 , the charge of the carbon atom in CH 2 turned to negative (−0.6572) from original a positive value (0.8612) in CO group, hence the nucleophilic attack by OH − became impossible.…”