Abstract

The reaction catalyzed by E. coli Pfk-2 presents a dual-cation requirement. In addition to that chelated by the nucleotide substrate, an activating cation is required to obtain full activity of the enzyme. Only Mn 2+ and Mg 2+ can fulfill this role binding to the same activating site but the affinity for Mn 2+ is 13-fold higher compared to that of Mg 2+. The role of the E190 residue, present in the highly conserved motif NXXE involved in Mg 2+ binding, is also evaluated in this behavior. The E190Q mutation drastically diminishes the kinetic affinity of this site for both cations. However, binding studies of free Mn 2+ and metal-Mant-ATP complex through EPR and FRET experiments between the ATP analog and Trp88, demonstrated that Mn 2+ as well as the metal-nucleotide complex bind with the same affinity to the wild type and E190Q mutant Pfk-2. These results suggest that this residue exert its role mainly kinetically, probably stabilizing the transition state and that the geometry of metal binding to E190 residue may be crucial to determine the catalytic competence. © 2010 Elsevier Inc. All rights reserved.