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J Biol Chem, Vol. 273, Issue 13, 7448-7456, March 27, 1998

Evidence for at Least Two Native Forms of Rabbit Muscle Adenylate Kinase in Equilibrium in Aqueous Solution

From the National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing 100101, China

The time course of 8-anilino-1-naphthalenesulfonic acid (ANS) binding to adenylate kinase (AK) is a biphasic process. The burst phase ends in the dead-time of the stopped-flow apparatus (about 15 ms), whereas the slow phase completes in about 10 min. A Job's plot tests of the binding stoichiometry demonstrates that there is one ANS binding site on AK, but only about 70% of the enzyme can rapidly bind with ANS, indicating that the conformation of native AK molecules is not homogeneous. Further kinetic analysis shows that the effects of ANS and substrates concentration on the burst and slow phase fluorescence building agree well with the multiple native forms mechanism. One form (denoted N₁) binds with ANS, whereas the other (denoted N₂) does not. ANS binding to N₁ results in a burst phase fluorescence increase, followed by the interconversion of N₂ to N₁, to give the slow phase ANS binding. Under urea denaturation conditions, N₂ is easily perturbed by urea and unfolds completely at low denaturant concentrations, whereas N₁ is relatively resistant to denaturation and unfolds at higher denaturant concentrations. The existence of multiple native forms in solution may shed some light on the interpretation of the enzyme catalytic mechanism.

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