THE ORIGINS AND CONSEQUENCES OF ASYMMETRY IN THE CHAPERONIN REACTION CYCLE

The binding of nucleotides and chaperonin-10 (cpn10) to the Escherichi a coli chaperonin-60 (cpn60) and their effect upon the molecular symme try has been examined both kinetically and at equilibrium. ATP binds t ightly and is hydrolysed on only one heptameric ring of the cpn60 tetr adecamer at a time, thus inducing asymmetry in the cpn60 oligomer even in the absence of cpn10. In the absence of cpn10 these seven ATP mole cules hydrolyse to form a cpn60:ADP(7) complex in which ADP is tightly bound (K-d = 2-7 mu M); further ADP binding to form a cpn60:ADP(14) c omplex is weak (K-1/2 = 2.3 mM). We conclude that symmetrical nucleoti de complexes (with 14 ATP or 14 ADPs) are unstable, demonstrating nega tive co-operativity between the rings. When cpn60 is mixed with cpn10 and ATP the resultant cpn60:ATP(7):cpn10 complex is formed rapidly (th e rate constant for cpn10 association is >4 x 10(7) M(-1) s(-1)) and b efore ATP is hydrolysed (k = 0.12 s(-1) per active subunit) to produce an extremely stable cpn60:ADP(7):cpn10 complex. This allows ATP assoc iation on the unoccupied ring and nucleotide asymmetry in the double t oroid is preserved. In ''trapping'' experiments, where the cpn60:ADP(7 ):cpn10 is challenged with ATP, cpn10 was observed to dissociate at a rate identical to that of steady-state ATP hydrolysis in the presence of cpn10 (k = 0.042 s(-1) per active subunit). The spontaneous decay o f cpn60:ADP(7):cpn10 and any of the major steady-state complexes, unde r conditions where free nucleotides had been removed, occurred at a ra te tenfold lower than ATP hydrolysis. Since the binding of the non-hyd rolysable analogue AMP-PNP was unable to induce dissociation of the co -chaperonin it was concluded that a transient state following Am hydro lysis is necessary for the rapid dissociation of cpn10, which occurs o nce in every cycle. Trapping experiments using sub-stoichiometric conc entrations of cpn10, relative to cpn60, show an unchanged rate of cpn1 0 exchange upon ATP hydrolysis, indicating that the formation of a sym metric, ''football''-shaped complex in which two molecules of the co-c haperonin are bound to cpn60, is not an obligatory intermediate in the exchange process.