Escherichia coli RNA polymerase is a metalloenzyme containing 2 g-atoms of tightly bound zinc per mol of enzyme. We have prepared RNA polymerase from E. coli cells grown in a zinc-depleted medium supplemented with cobalt(II) chloride. The purified enzyme contains 1.8-~2.2 g-atoms of cobalt per mol of enzyme with concomitant reduction in the zinc content. The cobalt-substituted enzyme is enzymatically as active as Zn-RNA polymerase on a variety of templates under standard assay conditions. These two enzymes are almost identical by such physical criteria as subunit composition, monomer-dimer equilibrium, and pH and temperature stabilities. They differ in that Co-RNA polymerase exhibits a visible absorption spectrum with two major peaks at 584 and 703 nm. Addition of nucleoside triphosphates selectively perturbs the 584-nm peak, whereas addition of a template analogue, d(pT)t0, affects both peaks. These spectral changes suggest that the tightly bound metal ions may directly or indirectly participate in binding of substrate or template to the enzyme. Biochemically, both enzymes are also very similar with respect to pH-activity profile, extrinsic metal require--Al variety of nucleotidyl transferases including DNA and RNA polymerases from both prokaryotic and eukaryotic sources have been shown to be zinc metalloenzymes (Slater et

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The structural determinants of substrate recognition in the human class IV, or , alcohol dehydrogenase (ADH) isoenzyme were examined through x-ray crystallography and site-directed mutagenesis. The crystal structure of ADH complexed with NAD

Summary Conformational dynamics has an established role in enzyme catalysis, but its contribution to ligand binding and specificity is largely unexplored. Here we used the Tiam1 PDZ domain and an engineered variant (QM PDZ) with broadened specificity to investigate the role of structure and conformational dynamics in molecular recognition. Crystal structures of the QM PDZ domain both free and bound to ligands showed structural features central to binding (enthalpy), while NMR-based methyl relaxation experiments and isothermal titration calorimetry revealed that conformational entropy contributes to affinity. In addition to motions relevant to thermodynamics, slower μs-ms switching was prevalent in the QM PDZ ligand binding site consistent with a role in ligand specificity. Our data indicate that conformational dynamics plays distinct and fundamental roles in tuning the affinity (conformational entropy) and specificity (excited-state conformations) of molecular interactions. More broadly, our results have important implications for the evolution, regulation and design of protein-ligand interactions.

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