Dissociation of yeast hexokinase by hydrostatic pressure

Abstract

The pressure-induced dissociation of the isozymes P1 and P2 of hexokinase was investigated by studies of the spectral shift of the intrinsic protein fluorescence and by the fluorescence polarization of dansyl conjugates. The free energy of association of the monomers at atmospheric pressure, Katm, was -14.2 kcal mol-1 at 20 degrees C and -11.4 kcal mol-1 at 0 degrees C. The positive enthalpy indicates that the association of the monomers is entropy-driven, overcoming the negative enthalpy of hydration of the subunit interfaces. At 0 degrees C and 1 bar, glucose stabilizes the association by -1.1 kcal mol-1 and the binding of both adenosine 5'-(beta, gamma-methylenetriphosphate) (AMPPCP) and glucose by an even larger amount, -1.34 kcal mol-1. Paradoxically, adenosine 5'-triphosphate (ATP), or AMPPCP, in the absence of glucose destabilizes the association by +0.34 kcal mol-1, while adenosine 5'-diphosphate (ADP) stabilizes it by -0.6 kcal mol-1. Comparison of dV0, the apparent standard volume of association, at different pHs and temperatures indicates that its value (115-160 mL mol-1) is strongly dependent upon the ionization of a group at the subunit interface with a pK near neutrality. Under dissociating pressures, trypsin action results in permanent dissociation of the dimer, confirming earlier observations of Colowick by less direct methods. The P1 and P2 enzymes differ in Katm and dV0 and markedly so in the effects of salt upon the stability of the dimer.(ABSTRACT TRUNCATED AT 250 WORDS)

Dissociation of peripheral protein‐membrane complexes by high pressure

“…That is, dissociation and reassociation showed no indication of the hysteresis that is displayed by many protein-protein interactions (Ruan & Weber, 1988. Thus, dissociation from membranes appeared to be an equilibrium process.…”

“…and KD(,t,) represent the dissociation equilibrium constant at pressure p and atmospheric pressure, respectively, R is the gas constant, Tis the temperature, and AV represents the volume change associated with complex formation (Deckmann et ai., 1985;Weber, 1987;Ruan & Weber, 1988). The primary contributors to volume changes are void volumes, which result from the imperfect interfacing of two macromolecular surfaces; electrostriction, the tighter packing of water molecules around an exposed charge (Heremans, 1982;Weber & Drickamer, 1983); exchange of more tightly packed polar (solvation) for nonpolar interactions; and lipid condensation in cases involving a membrane (Ceuterick et al, 1979;Deckmann et al, 1985;Wong, 1987).…”

“…The majority of previous studies of high pressure have involved the association/dissociation of oligomeric proteins (Payens & Heremans, 1969;Royer et al, 1986;Ruan & Weber, 1988;Silva & Weber, 1988). More re-…”

“…Changing the vesicle composition to phosphatidylethanolamine in place of PC gave higher affinity and decreased dissociation of the 32-kDa protein and SAP. Thus, the susceptibility of various peripheral membrane-binding proteins to high pressure dissociation was governed by both the KO (,,,) and the A 6 of the particular protein-membrane interaction, with the A 6 ' s of the dissociable proteins being relatively small (<I50 mL/mol).Keywords: annexin; calcium; factor V, light chain; high pressure; membrane-binding; phospholipid; protein kinase C; serum amyloid P component; vesicle; vitamin K-dependentThe effect of pressure on associated macromolecules is described by the expressionand KD(,t,) represent the dissociation equilibrium constant at pressure p and atmospheric pressure, respectively, R is the gas constant, Tis the temperature, and AV represents the volume change associated with complex formation (Deckmann et ai., 1985;Weber, 1987;Ruan & Weber, 1988). The primary contributors to volume changes are void volumes, which result from the imperfect interfacing of two macromolecular surfaces; electrostriction, the tighter packing of water molecules around an exposed charge (Heremans, 1982;Weber & Drickamer, 1983); exchange of more tightly packed polar (solvation) for nonpolar interactions; and lipid condensation in cases involving a membrane (Ceuterick et al, 1979;Deckmann et al, 1985;Wong, 1987).…”

“…Changes in protein tertiary structure typically are not observed at pressures less than 2.5 kbar (Heremans, 1982;Weber & Drickamer, 1983). Thus, high pressure can be a useful tool for identifying the type of forces involved in macromolecular interactions.The majority of previous studies of high pressure have involved the association/dissociation of oligomeric proteins (Payens & Heremans, 1969;Royer et al, 1986;Ruan & Weber, 1988;Silva & Weber, 1988). More re-…”