Pressure-induced perturbation on the active site of beta-amylase monitoredfrom the sulfhydryl reaction

We investigated the pressure effect on the conformation of P-amylase by mon itoring the chemical reaction of the unpaired cysteine. Sweet potato P-amyl ase is composed of four identical subunits, each of which contains six cyst eine residues. These residues are inert to 5,5'-dithiobis(2-nitrobenzoic ac id) (DTNB) in the native state due to steric hindrance. With the increase o f the pressure from 0.1 to 400 MPa, the reactivity of one cysteine out of s ix residues was enhanced. We have identified that the reacted cysteine resi due was Cys345 by the chemical cleavage at the reacted site. The reaction k inetics of Cys345 were pseudo-first-order, and the apparent rate constant w as increased from 0.001 to 0.05 min(-1) with the increase of pressure from 100 to 400 MPa. The activation volume of the reaction rate was calculated a s -24 +/- 2 mL/mol from the slope of the logarithmic plot of the pressure d ependence of the rate constant. Hysteresis was not evident in the change of intrinsic fluorescence during the cycle of compression and decompression b etween 0.1 and 400 MPa, indicating that the tetramer does not dissociate un der high pressure. This indicates that the enhancement of the reactivity of Cys345 was caused by the perturbation of local conformation under high pre ssure. The reaction of Cys345 was also enhanced by low concentrations of Gu HCl, suggesting the significant role of hydration-driven fluctuation in the pressure-induced enhancement of the reactivity,