Inactivation of creatine kinase by high pressure may precede dimer dissociation

Abstract

The effects of hydrostatic pressure on creatine kinase activity and conformation were investigated using either the high-pressure stopped-flow method in the pressure range 0.1±200 MPa for the activity determination, or the conventional activity measurement and fluorescence spectroscopy up to 650 MPa. The changes in creatine kinase activity and intrinsic fluorescence show a total or partial reversibility after releasing pressure, depending on both the initial value of the high pressure applied and on the presence or absence of guanidine hydrochloride. The study on 8-anilinonaphthalene-1-sulfonate binding to creatine kinase under high pressure indicates that the hydrophobic core of creatine kinase was progressively exposed to the solvent at pressures above 300 MPa. This data shows that creatine kinase is inactivated at low pressure, preceding both the enzyme dissociation and the unfolding of the hydrophobic core occurring at higher pressure. Moreover, in agreement with the recently published structure of the dimer, it can be postulated that the multistate transitions of creatine kinase induced both by pressure and guanidine denaturation are in direct relationship with the existence of hydrogen bonds which maintain the dimeric structure of the enzyme.Keywords: creatine kinase; dissociation; fluorescence; high-pressure fast kinetics; high-pressure inactivation.Cytoplasmic creatine kinase (CK, EC 2.7.3.2.), which catalyses the reversible transfer of phosphate from ATP to creatine, is an important enzyme for cellular energy metabolism. CK is typically found in cells that display high and variable rates of energy turnover. Its biological function and structural properties have been studied extensively [1,2]. The cytosolic enzyme from rabbit muscle (MM isoenzyme) is a dimer composed of two identical 43-kDa polypeptide chains of known sequence and it has been suggested that the enzyme must be in its dimeric form to be active [3]. The enzyme stability investigated by specific site-directed mutagenesis suggested that the dimeric state is required for monomer stabilization [4]. Conventional biophysical methods showed that during CK denaturation by guanidine hydrochloride or urea, inactivation occurs before significant conformational changes [5,6]. Using different physico-chemical approaches, equilibrium and kinetic studies of the CK unfolding-refolding induced by guanidine hydrochloride show two transitions, indicating the presence of at least one stable intermediate in the denaturation process at moderate guanidine concentrations [7]. Using the size-exclusion chromatography method, the presence of intermediates in equilibrium in the CK unfolding pathway has been also demonstrated, suggesting that it goes from dimeric to monomeric intermediates as a function of guanidine hydrochloride concentrations [8]. However, all these results came from unfolding studies using chemical denaturants, i.e. guanidine hydrochloride or urea. To rule out some possible direct effects of chemical denaturant on the unfolding, the pressure depende...

Pressure Denaturation of Phosphorylating Glyceraldehyde-3-Phosphate Dehydrogenase from Bacillus stearothermophilus

“…Therefore, as expected, denaturation of the GAPDH implies the loss of enzyme activity. This result is similar to that observed for yeast enolase where the monomers were inactive (6), but slightly different of the creatine kinase dissociation where the inactivation by high pressure may precede dimer dissociation (7).…”

“…Hydrostatic pressure is viewed as a gentle and reversible perturbant, making it a tool very useful to study the dissociation of oligomeric enzymes (5). Recently, in this laboratory, pressure has been used to dissociate yeast enolase and creatine kinase (6,7). Yeast GAPDH was shown to reversibly dissociate into the monomer under pressure (8).…”

Pressure Denaturation of the Yeast Prion Protein Ure2

“…This means that the transition observed for GdmCl denaturation corresponds to unfolding, with dissociation occurring silently within the unfolded baseline region. This therefore indicates that unlike many oligomeric proteins (15,26,28,29), high pressure does not induce dissociation of the Ure2 dimer, nor does it induce population of partially-folded intermediates under these experimental conditions. This study suggests that other methods that cause marginal destabilization of Ure2, such as urea (2) or pH (12), could be combined with hydrostatic pressure and thus might provide a useful means of investigating partially folded intermediates of Ure2.…”

“…A previous study showed that creatine kinase (CK), a dimeric protein of similar size and stability to Ure2, is also relatively resistant to pressure unfolding (26). The application of high pressure caused CK to adopt a partially folded intermediate state, closely resembling the molten globule state populated in moderate concentrations of GdmCl (27).…”

“…As hydrostatic pressure alone was insufficient to induce complete unfolding of Ure2, we investigated the effect of adding the chemical denaturant GdmCl (26). At atmospheric pressure, Ure2 is fully folded in 2 M GdmCl, fully denatured in 5 M GdmCl and the midpoint for the unfolding transition is 3 M GdmCl, as judged by a range of structural probes (2).…”