Candida yeasts are rarely infectious, but frequently cause life-threatening systemic
infections in patients immunocompromised by
AIDS or by immunosuppressive
therapeutics. The
secreted aspartic proteases (Saps) are known
virulence factors of pernicious Candida species. The most virulent, Candida albicans, possesses at least nine SAP genes, some of which are specifically expressed from cells with morphologies associated with virulence. Only one of these
proteases, Sap2, has been previously purified from yeast in sufficient quantities for enzymic studies. The other
enzymes are present in low amounts in yeast culture and are difficult to purify. As a consequence,
enzyme properties, including the substrate specificities, of all Saps are poorly studied. Therefore, four Saps that are known to be expressed in C. albicans, Sap1, Sap2, Sap3 and Sap6, were produced in Escherichia coli as recombinant
zymogens and purified in large quantities. These
proenzymes were autoactivated and purified as active
proteases. The enzymic properties including the substrate specificities at the P(1) and P(1)' sites were determined using a competitive hydrolysis method employing synthetic substrate mixtures. All four Saps cleave
peptide bonds between larger hydrophobic
amino acids, but these somewhat broad specificities differ in detail among the four
enzymes at both sites. At the P(1) site, Sap1, Sap2 and Sap6 prefer Phe while Sap3 prefers Leu. Positively charged
amino acids are also accommodated, especially by Sap2 and Sap3. The specificities at P(1)' are broader than at P(1) for all four
enzymes. Sap6 prefers Ala, whereas other Saps prefer Tyr. Acidic side chains are also accommodated at this site. Analysis of substrates with a hydrophobic
amino acid in P(1)' reveals that all the Saps possess a unique preference for Ala at this site. The observed differences of residue preferences among Saps may be utilized for the design of specific substrates and inhibitors.