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 N 1 ) binds with ANS, whereas the other (denoted N 2 ) does not. ANS binding to N 1 results in a burst phase fluorescence increase, followed by the interconversion of N 2 to N 1 , to give the slow phase ANS binding. Under urea denaturation conditions, N 2 is easily perturbed by urea and unfolds completely at low denaturant concentrations, whereas N 1 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.It has been accepted that a globular protein in its native state adopts a single, well defined conformation. However, this concept has been challenged by several reports that some proteins may exist in more than one distinct folded form in equilibrium. Evidence for distinguishing multiple native forms of staphylococcal nuclease has come from electrophoretic and NMR studies (1-6). For calbindin D 9K , evidence of multiple forms came not only from NMR studies, but also from x-ray crystal structure (7,8).Adenylate kinase (AK 1 ; EC 2.7.4.3) catalyzes the phosphoryl transfer reaction: MgATP ϩ AMP i MgADP ϩ ADP (9, 10). The enzyme contains two distinct nucleotide binding sites: the MgATP site, which binds MgATP and MgADP, and the AMP site, which is specific for AMP and uncomplexed ADP. The substrate-induced conformation changes in AK have been the subject of a number of investigations (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Based on the comparison of AK crystal structures representing the enzyme in different ligand forms, apo-form (from pig muscle), enzyme-AMP binary complex (from beef heart mitochondrial matrix), and enzyme-AP 5 A complex (from Escherichia coli), Schulz and co-workers (15, 18) suggested that AK undergoes large structural changes upon substrates binding. These conformational changes can be subdivided into two steps; the first change, corresponding to binding to AMP, only involves the displacement of the small ␣-helical domain (residues 30 -59 in E. coli AK), whereas the second change, occurring with additional binding of substrate ATP, mainly involves the displacement of the LID domain (residues 122-159 in E. coli AK). However, it is not clear whether the enzyme achieves its catalytic conformation only upon substrate binding or whether the above confo...