Cryoenzymology of porcine pepsin

Physical and kinetic properties of porcine pepsin have been examined in aqueous methanol solvents at temperatures below ambient to seek evidence for covalent intermediates in the catalyzed hydrolysis of good substrates. It was first demonstrated that aqueous methanol cryosolvents have no significant deleterious effects upon this protein. The addition of methanol does lead to a drastic reduction in the midpoint of the thermal melting curve of pepsin. This could account for rate reductions previously observed in catalysis by this enzyme. This effect is lessened by the addition of active-site ligands including substrates and is fully reversible upon dilution into aqueous solution. Two substrates were chosen which have chromophoric groups on opposite sides of the scissile peptide bond. The UV spectral changes from hydrolysis of Pro-Thr-Glu-Phe-(NO2)Phe-Arg-Leu and the fluorescence spectral changes from hydrolysis of DNS-Ala-Ala-Phe-Phe-OP4P+-CH3 were studied at temperatures down to -60 degrees C. The resulting Arrhenius plots were linear in the region where pepsin exists in the native state with downward curvature exhibited at higher temperatures where the reversible denaturation occurs. No "burst" reactions were observed with either substrate. In addition, efforts at trapping intermediates by low-temperature denaturation and precipitation have provided no evidence for covalent intermediates on the reaction pathway. Although this evidence is negative, we cannot rule out the possibility of the formation of covalent intermediates following an initial rate-limiting step.