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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Twelve isomers formed by the reaction of monoamminechromium(III) with ATP have been synthesized. Isomerism in this system results from chirality around the beta-phosphorus of the ATP, the position of the ammonia ligand, the relative orientation of the ammonia and the AMP, and the presence of ring-puckering conformers. By using chromatography on cross-linked cycloheptaamylose, reverse-phase C-18 HPLC, and cation-exchange FPLC, these isomers have been separated and purified. Their structures have been identified by (1) cleavage by periodate, followed by elimination in the presence of diethylenetriamine and subsequent phosphate insertion to give lambda, delta, or meso facial monoamminechromium tripolyphosphate with molar ellipticities of +240, -240, or 0 deg cm2 dmol-1 at 550 nm, respectively, (2) cleavage by nucleotide pyrophosphatase to give meridional or facial monoamminechromium pyrophosphate, (3) spectral data, and (4) rates of interconversion of isomers. All possible isomers are seen except those with ammonia syn to AMP. Since the substitution of ammonia for water in the inner coordination sphere appears to diminish affinity for enzymes when the ammonia is in contact with the protein but not when it faces the solvent, these isomers are useful for mapping of enzyme active sites. Their use as probes of enzyme structure is illustrated by their behavior with yeast hexokinase.