Density functional and continuum dielectric theories have been combined to calculate molecular properties such as hydration enthalpies, redox potentials, and absolute pK a values of transition metal cations in solution. The discrete cluster model, which is treated explicitly by density functional theory, includes six waters in the first hydration shell and another twelve waters in the second shell. The solvent reaction field is obtained from a finite-difference solution to the Poisson−Boltzmann equation and is coupled to the nonlocal density functional calculation in a self-consistent way. The calculated hydration enthalpies are 409, 1073, 431, and 1046 kcal/mol for Mn2+, Mn3+, Fe2+, and Fe3+, respectively, comparing fairly well to the experimental measurements of 440, 1087, 465, and 1060 kcal/mol. The calculated redox potentials for the Mn2+/Mn3+ and Fe2+/Fe3+ pairs are 1.59 and 1.06 V, respectively, in good agreement with the experimental values of 1.56 and 0.77 V. The computed absolute pK a values, 14.0, −6.5, 9.0, and −4.0 for Mn2+, Mn3+, Fe2+, and Fe3+, respectively, deviate significantly from the experimental results of 10.6, 0.1, 9.5, and 2.2 but show the proper behavior with changes in oxidation state and metal type. The calculated redox potentials and pK a values appear to converge toward the experimental data with increasing size of the cluster models. For such highly charged cations, the second hydration shell in the cluster model is indispensable, since this buffer shell retains strong hydrogen bonds and electron transfer between the inner and outer shells as well as the solute−solvent dispersion interaction.

The enzyme Cu, Zn superoxide dismutase (SOD) protects against oxidative damage by dismuting the superoxide radical O2-. to molecular oxygen and hydrogen peroxide at the active-site Cu ion in a reaction that is rate-limited by diffusion and enhanced by electrostatic guidance. SOD has evolved to be one of the fastest enzymes known (V(max) approximately 2 x 10(9) M-1 s-1). The new crystal structures of human SOD show that amino-acid site chains that are implicated in electrostatic guidance (Glu 132, Glu 133 and Lys 136) form a hydrogen-bonding network. Here we show that site-specific mutants that increase local positive charge while maintaining this orienting network (Glu----Gln) have faster reaction rates and increased ionic-strength dependence, matching brownian dynamics simulations incorporating electrostatic terms. Increased positive charge alone is insufficient: one charge reversal (Glu----Lys) mutant is slower than the equivalent charge neutralization (Glu----Gln) mutant, showing that the newly introduced positive charge disrupts the orienting network. Thus, electrostatically facilitated diffusion rates can be increased by design, provided the detailed structural integrity of the active-site electrostatic network is maintained.

Some genital human papillomavirus (HPV) types, such as 16 and 18, are highly associated with malignant cervical tumors while others, such as HPV 6, are only rarely found in these malignancies. The E7 oncoproteins of HPV 6, 16 and 18 each have a 17 amino acid region with striking homology to adenovirus E1a and SV40 LT. E1a, LT and the E7 oncoprotein of HPV16 all bind the cellular Rb protein in vitro, and for E1a and LT this region of homology contains sequences essential for interaction with Rb. We have now found that in HPV 16 E7 this region (amino acids 21‐37) contains two separate biochemical activities, each of which contributes to E7‐mediated transformation. Rb binding was localized to the N terminus of this region, while the C terminus was shown to serve as a substrate for casein kinase (CK) II, which phosphorylated serine‐31 and serine‐32. Replacement of the two serines by non‐phosphorylatable amino acids led to a reduction in transforming activity and abolished phosphorylation but did not affect Rb binding. Rb binding and CK II phosphorylation were also examined for the E7 proteins of HPV 6 and HPV 18. HPV 16 and 18 E7 bound similar amounts of Rb, but HPV 6 E7 consistently bound less. Phosphorylation rates also varied, with HPV 18 E7 being 2‐fold faster than HPV 16 E7, which in turn was 2‐fold faster than HPV 6 E7. We conclude that Rb binding and phosphorylation of E7 by CKII are independent activities which are required for efficient transformation by E7 and that these activities correlate directly with the relative oncogenic potential of these viruses.

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