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Residue 75 on the flap, a beta hairpin loop that partially covers the active site cleft, is tyrosine in most members of the aspartic proteinase family. Site-directed mutagenesis was carried out to investigate the functional role of this residue in Rhizomucor pusillus pepsin, an aspartic proteinase with high milk-clotting activity produced by the fungus Rhizomucor pusillus. A set of mutated enzymes with replacement of the amino acid at position 75 by 17 other amino acid residues except for His and Gly was constructed and their enzymatic properties were examined. Strong activity, higher than that of the wild-type enzyme, was found in the mutant with asparagine (Tyr75Asn), while weak but distinct activity was observed in Tyr75Phe. All the other mutants showed markedly decreased or negligible activity, less than 1/1000 of that of the wild-type enzyme. Kinetic analysis of Tyr75Asn using a chromogenic synthetic oligopeptide as a substrate revealed a marked increase in kcat with slight change in K(m), resulting in a 5.6-fold increase in kcat/K(m). When differential absorption spectra upon addition of pepstatin, a specific inhibitor for aspartic proteinase, were compared between the wild-type and mutant enzymes, the wild-type enzyme and Tyr75Asn, showing strong activity, had spectra with absorption maxima at 280, 287 and 293 nm, whereas the others, showing decreased or negligible activity, had spectra with only two maxima at 282 and 288 nm. This suggests a different mode of the inhibitor binding in the latter mutants. These observations suggest a crucial role of the residue at position 75 in enhancing the catalytic efficiency through affecting the mode of substrate-binding in the aspartic proteinases.

protein engineering, design and selection

Involvement of a residue at position 75 in the catalytic mechanism of a fungal aspartic proteinase, <i>Rhizomucor pusilus</i> pepsin. Replacement of tyrosine 75 on the flap by asparagine enhances catalytic efficiency

Involvement of a residue at position 75 in the catalytic mechanism of a fungal aspartic proteinase, Rhizomucor pusilus pepsin. Replacement of tyrosine 75 on the flap by asparagine enhances catalytic efficiency