SUPRAMOLECULAR SELF-ASSEMBLY OF ESCHERICHIA-COLI GLUTAMINE-SYNTHETASE- EFFECTS OF PRESSURE AND ADENYLYLATION STATE ON DODECAMER STACKING

Escherichia coli glutamine synthetase is a dodecamer of identical subu nits, consisting of two face-to-face hexameric rings. The enzymatic ac tivity of GS is regulated by covalent attachment of an adenylyl group to each subunit, at the edge of the ring structure (Tyr-397). In the p resence of Zn2+, Cu2+, Co2+, and other divalent metal ions, the free d odecamers self-organize into protein tabules [Miller et al. (1974) Arc h, Biochem. Biophys. 163, 155-171]. Here, the temperature dependence a nd pressure dependence of the kinetics of Zn2+-induced self-assembly o f GS tubules have been determined for the adenylylated and unadenylyla ted GS. The adenylylated enzyme exhibits a bimolecular rate constant f or Zn2+-induced stacking that is 3-fold lower than for the unadenylyla ted GS at temperatures ranging from 0 to 25 degrees C. The enthalpy of activation, Delta H double dagger, far both adenylylated and unadenyl ylated GS increases from approximately 10 kcal/mol of dodecamer interf ace to 20 kcal/mol of dodecamer interface upon addition of 125 mM KCl to the reaction buffer. The Delta H double dagger values for adenylyla ted and unadenylylated GS are nearly identical, at each concentration of KCl, suggesting that entropic factors are responsible for the diffe rences in rate of stacking for these forms of GS. Hydrostatic pressure markedly inhibits the stacking reaction for both adenylylated and una denylylated GS. The activation volumes, Delta V double dagger(a), for stacking are increased from approximately 50 mL/mol of dodecamer inter face in the absence of KCl to approximately 65 mL/mol of dodecamer int erface in the presence of 125 mM KCl. The kinetics of pressure-induced disassembly of GS tubules were also examined. ''Pressure-jump'' exper iments with preformed GS tubules indicate that rates of dissociation a t atmospheric pressure, in the presence of 200 mu M Zn2+ and 125 mM KC l, are 1.1 x 10(-5) s(-1) and 1.9 x 10(-5) s(-1) for the unadenylylate d and adenylylated GS, respectively. The Delta V double dagger(d) valu es for disassembly increase from approximately -63 mL/mol of dodecamer interface in the absence of KCl to approximately -55 mL/mol of dodeca mer interface in the presence of 125 mM KCl. These results suggest tha t the encounter complex formed between dodecamers, en route to the sta cked complex, is not extensively desolvated.; Furthermore, the experim entally determined rate constants for the forward and reverse reaction s have been used to calculate apparent equilibrium dissociation consta nts. These Values range from 1.3 x 10(-10) M(-1) to 8.0 x 10(-9) M(-1) , and they are increased by adenylylation and by addition of KCl.