Use of hydrostatic pressure to produce 'native' monomers of yeast enolase

Abstract

The effects of hydrostatic pressure on yeast enolase have been studied in the presence of 1 mM Mn2+. When compared with apo-enolase, and Mg-enolase, the Mn-enzyme differs from the others in three ways. Exposure to hydrostatic pressure does not inactivate the enzyme. If the experiments are performed in the presence of 1 mM Mg2+, or with apo-enzyme, the enzyme is inactivated [Kornblatt, M.J., Lange R., Balny C. (1998) Eur. J. Biochem 251, 775-780]. The UV spectra of the high pressure forms of the Mg 2+- and apo-forms of enolase are identical and distinct from the spectrum of the form obtained in the presence of 1 mM Mn2+; this suggests that Mn2+ remains bound to the high pressure form of enolase. With Mn-enolase, the various spectral changes do not occur in the same pressure range, indicating that multiple processes are occurring. Pressure experiments were performed as a function of [Mn2+] and [protein]. One of the changes in the UV spectra shows a dependence on protein concentration, indicating that enolase is dissociating into monomers. The small changes in the UV spectrum and the retention of activity lead to a model in which enolase, in the presence of high concentrations of Mn2+, dissociates into native monomers; upon release of pressure, the enzyme is fully active. Although further spectral changes occur at higher pressures, there is no inactivation as long as Mn2+ remains bound. We propose that the relatively small and polar nature of the subunit interface of yeast enolase, including the presence of several salt bridges, is responsible for the ability of hydrostatic pressure to dissociate this enzyme into monomers with a native-like structure.