EFFECT OF 2 POLAR ORGANIC-AQUEOUS SOLVENT SYSTEMS ON THE STRUCTURE-FUNCTION-RELATIONSHIPS OF PROTEASES .2. CHYMOSIN AND MUCOR-MIEHEI PROTEINASE

The kinetics of chymosin and Mucor miehei proteinase (Mmp) catalyzed p roteolysis of sodium (Na) caseinate, as well as their intrinsic struct ural properties, were examined in two polar organic-aqueous solvent sy stems. 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 para meters for the hydrolysis of Na-caseinate by chymosin showed that, rel ative to standard buffer, the K(m) in both 5 % EtOH in standard buffer and 5 % ACN in standard buffer increased significantly (p less-than-o r-equal-to 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 o bserved in V(max) in 5% EtOH (12.8 mumoles/min. mg) or 5 % ACN (11. 8 mumoles/min. mg) compared with standard buffer (12.8 mumoles/min.mg) r esulting in V(max)/K(m) values reduced to a similar extent in both org anic solvents. For Mmp, a glycoprotein, K(m) increased (p less-than-or -equal-to 0. 05) in 5 % ACN in standard buffer (5.3 mg/ml, yet it decr eased (p less-than-or-equal-to 0. 05) in 5 % EtOH in standard buffer ( 3.1 mg/ml) compared with standard buffer (4.2 mg/ml). The solvent-indu ced decrease in V(max) for Mmp was somewhat larger in 5 % EtOH in stan dard buffer than 5 % ACN in standard buffer, from 17.3 mumoles/min. mg (standard buffer) to 14.0 mumoles/min. mg (p less-than-or-equal-to 0. 05) in 5 % EtOH in standard buffer but was not significantly differen t (p > 0.05) in 5 % ACN in standard buffer (15.7 mumoles/min mg). For both chymosin and Mmp, changes in kinetic parameters appeared to corre spond with solvent-induced structural changes as evidenced by near-UV circular dichroism (CD) spectroscopy, higher K(m) corresponded to a hi gher ellipticity in the near-UV CD spectra (240-320 nm), which may ind icate a decreased protein flexibility. The fact that chymosin and Mmp behaved differently in these organic solvents indicates that factors o ther than polarity of the media may also have been involved in this ph enomenon since both 5 % EtOH in standard buffer and 5 % ACN in standar d buffer solutions were of the same polarity based on E(T)(30) scale. Different specificities for Na-caseinate hydrolysis between chymosin a nd Mmp, as well as altered specificities of both enzymes for the subst rate caused by organic solvents, were demonstrated in SDS-PA GE peptid e maps as differences in banding patterns. Differential scanning calor imetric studies on chymosin and Mmp in the two solvent systems showed destabilization (lowered temperature of denaturation) of the enzymes i n both 5 % EtOH in standard buffer and 5 % ACN in standard buffer rela tive to standard buffer.