EFFECT OF TWO POLAR ORGANIC-AQUEOUS SOLVENT SYSTEMS ON THE STRUCTURE-FUNCTION RELATIONSHIPS OF PROTEASES II. CHYMOSIN AND MUCOR MIEHEI PROTEINASE

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The kinetics of chymosin and Mucor miehei proteinase (Mmp) catalyzed proteolysis of sodium (Na) caseinate, as well as their intrinsic structural properties, were examined in two polar organic-aqueous solvent systems. Chymosin and Mmp in standard buffer (10 mM phosphate, pH 6.0), 5% (v/v) ethanol (EtOH) in standard buffer and 5% (v/v) acetonitrile (ACN) in standard buffer were used in the investigation. Kinetic parameters for the hydrolysis of Na-caseinate by chymosin showed that, relative to standard buffer, the Km in both 5% EtOH in standard buffer and 5% ACN in standard buffer increased significantly (p ≤ 0.05) to nearly the same level from 4.8 mg/ml to 7.5 mg/ml and 7.7 mg/ml, respectively. No significant changes (p > 0.05) were observed in Vmax in 5% EtOH (12.8 μmoles/min.mg) or 5% ACN (11.8 μmoles/min.mg) compared with standard buffer (12.8 μmoles/min.mg) resulting in Vmax/Km values reduced to a similar extent in both organic solvents. For Mmp, a glycoprotein, Km increased (p ≤ 0.05) in 5% ACN in standard buffer (5.3 mg/ml, yet it decreased (p ≤ 0.05) in 5% EtOH in standard buffer (3.1 mg/ml) compared with standard buffer (4.2 mg/ml). The solvent-induced decrease in Vmax for Mmp was somewhat larger in 5% EtOH in standard buffer than 5% ACN in standard buffer, from 17.3 μmoles/min.mg (standard buffer) to 14.0 μmoles/min.mg (p ≤ 0.05) in 5% EtOH in standard buffer but was not significantly different (p > 0.05) in 5% ACN in standard buffer (15.7 μmoles/min.mg). For both chymosin and Mmp, changes in kinetic parameters appeared to correspond with solvent-induced structural changes as evidenced by near-UV circular dichroism (CD) spectroscopy; higher Km corresponded to a higher ellipticity in the near-UV CD spectra (240–320 nm), which may indicate a decreased protein flexibility. The fact that chymosin and Mmp behaved differently in these organic solvents indicates that factors other than polarity of the media may also have been involved in this phenomenon since both 5% EtOH in standard buffer and 5% ACN in standard buffer solutions were of the same polarity based on ET(30) scale. Different specificities for Na-caseinate hydrolysis between chymosin and Mmp, as well as altered specificities of both enzymes for the substrate caused by organic solvents, were demonstrated in SDS-PAGE peptide maps as differences in banding patterns. Differential scanning calorimetric studies on chymosin and Mmp in the two solvent systems showed destabilization (lowered temperature of denaturation) of the enzymes in both 5% EtOH in standard buffer and 5% ACN in standard buffer relative to standard buffer.