Dissecting a bimolecular process of MgATP²- binding to the chaperonin GroEL

Although allosteric transitions of GroEL by MgATP(2)(-) have been widely studied, the initial bimolecular step of MgATP(2-) binding to GroEL remains unclear. Here, we studied the equilibrium and kinetics of MgATP(2)(-) binding to a variant of GroEL, in which Tyr485 was replaced by tryptophan, via isothermal titration calorimetry (ITC) and stopped-flow fluorescence spectroscopy. In the absence of K(+) at 4-5 °C, the allosteric transitions and the subsequent ATP hydrolysis by GroEL are halted, and hence, the stopped-flow fluorescence kinetics induced by rapid mixing of MgATP(2)(-) and the GroEL variant solely reflected MgATP(2)(-) binding, which was well represented by bimolecular noncooperative binding with a binding rate constant, k(on), of 9.14×10(4) M(-1) s(-1) and a dissociation rate constant, k(off), of 14.2 s(-1), yielding a binding constant, K(b) (=k(on)/k(off)), of 6.4×10(3) M(-1). We also successfully performed ITC to measure binding isotherms of MgATP(2)(-) to GroEL and obtained a K(b) of 9.5×10(3) M(-1) and a binding stoichiometric number of 6.6. K(b) was thus in good agreement with that obtained by stopped-flow fluorescence. In the presence of 10-50 mM KCl, the fluorescence kinetics consisted of three to four phases (the first fluorescence-increasing phase, followed by one or two exponential fluorescence-decreasing phases, and the final slow fluorescence-increasing phase), and comparison of the kinetics in the absence and presence of K(+) clearly demonstrated that the first fluorescence-increasing phase corresponds to bimolecular MgATP(2)(-) binding to GroEL. The temperature dependence of the kinetics indicated that MgATP(2)(-) binding to GroEL was activation-controlled with an activation enthalpy as large as 14-16 kcal mol(-1).