The beta-subunits of the voltage-gated potassium (Kv) channels modulate the kinetics and the gating of Kv channels and assists in channel trafficking and membrane localization. These proteins are members of the AKR6 family. They share a common (alpha/beta)(8) barrel structural fold and avidly bind pyridine nucleotides. Low catalytic activity has been reported for these proteins. Kinetic studies with rat Kvbeta2 revealed that the chemical step is largely responsible for the rate-limitation but nucleotide exchange could also contribute to the overall rate. Herein we report our investigations on the kinetics of cofactor exchange using nucleotide-free preparations of Kvbeta2. Kinetic traces measuring quenching of Kvbeta2 fluorescence by NADP(+) were consistent with a two-step binding mechanism which includes rapid formation of a loose enzyme:cofactor complex followed by a slow conformational rearrangement to form a tight final complex. Closing of the nucleotide enfolding loop, which in the crystal structure folds over the bound cofactor, provides the structural basis for this rearrangement. The rate of the loop opening required to release the cofactor is similar for NADPH and NADP(+) (0.9 min(-1)) and is of the same order of magnitude as the rate of the chemical step estimated previously from kinetic studies with 4-nitrobenzaldehyde (0.3-0.8 min(-1), [S.M. Tipparaju, O.A. Barski, S. Srivastava, A. Bhatnagar, Catalytic mechanism and substrate specificity of the beta-subunit of the voltage-gated potassium channel, Biochemistry 47 (2008) 8840-8854]). Binding of NADPH is accompanied by a second conformational change that might be responsible for a 4-fold higher affinity observed with the reduced cofactor and the resulting difficulty in removing bound NADPH from the protein. These data provide evidence that nucleotide exchange occurs on a seconds-to-minutes time scale and set the upper limit for the maximal possible rate of catalysis by Kvbeta2. Slow cofactor exchange is consistent with the role of the beta-subunit as a metabolic sensor implicated in tonic regulation of potassium currents.