The reaction of reduced NO synthase (NOS) with molecular oxygen was studied at ؊30°C. In the absence of substrate, the complex formed between ferrous NOS and O 2 was sufficiently long lived for a precise spectroscopic characterization. This complex displayed similar spectral characteristics as the oxyferrous complex of cytochrome P450 ( max ‫؍‬ 416.5 nm). It then decomposed to the ferric state. The oxidation of the flavin components was much slower and could be observed only at temperatures higher than ؊20°C.

Elevated hydrostatic pressure has been used to increase catalytic activity and thermal stability of α‐chymotrypsin (CT). For an anilide substrate, characterized by a negative value of the reaction activation volume (ΔV≠), an increase in pressure at 20°C results in an exponential acceleration of the hydrolysis rate catalyzed by CT reaching a 6.5‐fold increase in activity at 4700 atm (4.7 kbar). Due to a strong temperature dependence of ΔV≠, the acceleration effect of high pressure becomes more pronounced at high temperatures. For example, at 50°C, under a pressure of 3.6 kbar, CT shows activity which is more than 30 times higher than the activity at normal conditions (20°C, 1 atm). At pressures of higher than 3.6 kbar, the enzymatic activity is decreased due to a pressure‐induced denaturation. Elevated hydrostatic pressure is also efficient for increasing stability of CT against thermal denaturation. For example, at 55°C, CT is almost instantaneously inactivated at atmospheric pressure, whereas under a pressure of 1.8 kbar CT retains its anilide‐hydrolyzing activity during several dozen minutes. Additional stabilization can be achieved in the presence of glycerol, which is most effective for protection of CT at an intermediate concentration of 40% (v/v). There has been observed an additivity in stabilization effects of high pressure and glycerol: thermal inactivation of pressure‐stabilized CT can be decelerated in a supplementary manner by addition of 40% (v/v) glycerol. The protection effect of glycerol on the catalytic activity and stability of CT becomes especially pronounced when both extreme factors of temperature and pressure reach critical values. For example, at approximately 55°C and 4.7 kbar, enzymatic activity of CT in the presence of 40% (v/v) glycerol is severalfold higher than in aqueous buffer. The results of this study are discussed in terms of the hypotheses which explain the action of external and medium effects on protein structure, such as preferential hydration and osmotic pressure. © 1996 John Wiley & Sons, Inc.

To investigate the characteristics of the postulated carboxy terminal chain-folding initiation site in bovine pancreatic ribonuclease A (RNase A) (residues 106-118), important in the early stages of the folding pathway, we have engineered by site-directed mutagenesis a set of 14 predominantly conservative hydrophobic variants of the protein. The stability of each variant has been compared by pressure and temperature-induced unfolding, monitored by fourth derivative UV absorbance spectroscopy. Apparently simple two-state, reversible unfolding transitions are observed, suggesting that the disruption of tertiary structure of each protein at high pressure or temperature is strongly cooperative. Within the limits of the technique, we are unable to detect significant differences between the two processes of denaturation. Both steady-state kinetic parameters for the enzyme reaction and UV CD spectra of each RNase A variant indicate that truncation of hydrophobic side chains in this region has, in general, little or no effect on the native structure and function of the enzyme. Furthermore, the decreases in free energy of unfolding upon pressure and thermal denaturation of all the variants, particularly those modified at residues 106 and 108, suggest that the hydrophobic residues and side chain packing interactions of this region play an important role in maintaining the conformational stability of RNase A. We also demonstrate the potential of Tyr115 replacement by Trp as a non-destabilizing fluorescence probe of conformational changes local to the region.