Enzymes efficiently catalyze reactions by stabilizing inherently unstable transition states. For cysteine proteases, part of the stabilization is provided by a region of the enzyme termed the oxyanion hole. Site-directed mutagenesis has been used to investigate further the role of the oxyanion hole of papain in the binding of putative transition state analog inhibitors of cysteine proteases. The dissociation constants Kiobs for inhibition of wild-type and mutant enzymes (Gln19Ala, Glnl9Glu , and Glnl9His) by the aldehyde Ac-Phe-Gly-CHO and the nitrile MeOCO-Phe-Gly-CN have been determined in the pH range 3.5-9.0. For the peptide nitrile inhibitor, mutation of Gln19 was found to cause important increases in Kiobs, and thioimidate adducts with the papain mutants Gln19Ala and Gln19Glu are less stable by 1.4-2.4 kcal/mol. However, for the peptide aldehyde inhibitor, the mutations resulted in a small but significant increase in stability of the tetrahedral hemithioacetal adduct (0.4-1.2 kcal/mol). In that respect, the hemithioacetal formed between papain and a peptide aldehyde cannot be considered a good model of the transition state for cysteine protease-catalyzed reactions. The influence of the mutations on the pH dependency of inhibition also indicates that with respect to oxyanion hole interaction, the inhibition of papain by peptide nitriles is a process closer to that of substrate hydrolysis than is the inhibition by the corresponding peptide aldehydes. The nature of the intermediates and transition states in hydrolysis reactions catalyzed by cysteine proteases, as well as the use of enzyme-inhibitor adducts as their models, is discussed.