Cindy L. Fisher

The interdependence of protein surface topography and bound water molecules revealed by surface accessibility and fractal density measures

To characterize water binding to proteins, which is fundamental to protein folding, stability and activity, the relationships of 10,837 bound water positions to protein surface shape and residue type were analyzed in 56 high-resolution crystallographic structures. Fractal atomic density and accessibility algorithms provided an objective characterization of deep grooves in solvent-accessible protein surfaces. These deep grooves consistently had approximately the diameter of one water molecule, suggesting that deep grooves are formed by the interactions between protein atoms and bound water molecules. Protein surface topography dominates the chemistry and extent of water binding. Protein surface area within grooves bound three times as many water molecules as non-groove surface; grooves accounted for one-quarter of the total surface area yet bound half the water molecules. Moreover, only within grooves did bound water molecules discriminate between different side-chains. In grooves, main-chain surface was as hydrated as that of the most hydrophilic side-chains, Asp and Glu, whereas outside grooves all main and side-chains bound water to a similar, and much decreased, extent. This identification of the interdependence of protein surface shape and hydration has general implications for modelling and prediction of protein surface shape, recognition, local folding and solvent binding.

Computational, pulse-radiolytic, and structural investigations of lysine-136 and its role in the electrostatic triad of human Cu,Zn superoxide dismutase.

Key charged residues in Cu,Zn superoxide dismutase (Cu,Zn SOD) promote electrostatic steering of the superoxide substrate to the active site Cu ion, resulting in dismutation of superoxide to oxygen and hydrogen peroxide. Lys‐136, along with the adjacent residues Glu‐132 and Glu‐133, forms a proposed electrostatic triad contributing to substrate recognition. Human Cu,Zn SODs with single‐site replacements of Lys‐136 by Arg, Ala, Gln, or Glu or with a triple‐site substitution (Glu‐132 and Glu‐133 to Gln and Lys‐136 to Ala) were made to test hypotheses regarding contributions of these residues to Cu,Zn SOD activity. The structural effects of these mutations were modeled computationally and validated by the X‐ray crystallographic structure determination of Cu,Zn SOD having the Lys‐136‐to‐Glu replacement. Brownian dynamics simulations and multiple‐site titration calculations predicted mutant reaction rates as well as ionic strength and pH effects measured by pulse‐radiolytic experiments. Lys‐136‐to‐Glu charge reversal decreased dismutation activity 50% from 2.2 × 109 to 1.2 × 109 M−1 s−1 due to repulsion of negatively charged superoxide, whereas charge‐neutralizing substitutions (Lys‐136 to Gln or Ala) had a less dramatic influence. In contrast, the triple‐mutant Cu,Zn SOD (all three charges in the electrostatic triad neutralized) surprisingly doubled the reaction rate compared with wild‐type enzyme but introduced phosphate inhibition. Computational and experimental reaction rates decreased with increasing ionic strength in all of the Lys‐136 mutants, with charge reversal having a more pronounced effect than charge neutralization, implying that local electrostatic effects still govern the dismutation rates. Multiple‐site titration analysis showed that deprotonation events throughout the enzyme are likely responsible for the gradual decrease in SOD activity above pH 9.5 and predicted a pKa value of 11.7 for Lys‐136. Overall, Lys‐136 and Glu‐132 make comparable contributions to substrate recognition but are less critical to enzyme function than Arg‐143, which is both mechanistically and electrostatically essential. Thus, the sequence‐conserved residues of this electrostatic triad are evidently important solely for their electrostatic properties, which maintain the high catalytic rate and turnover of Cu,Zn SOD while simultaneously providing specificity by selecting against binding by other anions. Proteins 29:103–112, 1997.

Probing the structural basis for enzyme-substrate recognition in Cu,Zn superoxide dismutase.

A full understanding of enzyme-substrate interactions requires a detailed knowledge of their structural basis at atomic resolution. Crystallographic and biochemical data have been analyzed with coupled computational and computer graphic approaches to characterize the molecular basis for recognition of the superoxide anion substrate by Cu,Zn superoxide dismutase (SOD). Detailed analysis of the bovine SOD structure aligned with SOD sequences from 15 species provides new results concerning the significance and molecular basis for sequence conservation. Specific roles have been assigned for all 23 invariant residues and additional residues exhibiting functional equivalence. Sequence invariance is dominated by 15 residues that form the active site stereochemistry, supporting a primary biological function of superoxide dismutation. Using data from crystallographic structures and site-directed mutants, we are testing the role of individual residues in the active site channel, including (in human SOD) Glu 132, Glu 133, Lys 136, Thr 137, and Arg 143. Electrostatic calculations incorporating molecular flexibility suggest that the region of positive electrostatic potential in and over the active site channel above the Cu ion sweeps through space during molecular motion to enhance the facilitated diffusion responsible for the enzymes rapid catalytic rate.

MECHANISM AND ATOMIC STRUCTURE OF SUPEROXIDE DISMUTASE

The active site Cu ion in Cu,Zn superoxide dismutase is alternately oxidized and reduced during the enzymatic dismutation of superoxide to hydrogen peroxide and molecular oxygen. For oxidized Cu,Zn superoxide dismutase, an atomic structure has been determined for the human enzyme at 2.5 A resolution. The resolution of the bovine enzyme structure has been extended to 1.8 A. Atomic resolution data has been collected for reduced and inhibitor-bound Cu,Zn superoxide dismutases, and the interpretation of the electron density difference maps is in progress. The geometry and molecular surfaces of the active sites in these structures, together with biochemical data, suggest a specific model for the enzyme mechanism. Similarities in the active site geometry of the Mn and Fe superoxide dismutases with the Cu,Zn enzyme suggest that dismutation in these enzymes may follow a similar mechanism.