Structural studies of the catalytic mechanisms of two superfast metalloenzymes: The carbonic anhydrases and manganese superoxide dismutases

The utilization of metals in biological enzymes is ubiquitous in the diverse kingdoms of life. These metals, on their own are inactive, but when incorporated into a protein allow for the enhancement a myriad of chemical reactions. To understand how an enzyme functions it is necessary to gain detailed knowledge of its active site structure and how this correlates to catalytic efficiency. Additionally, a knowledge of the interactions between enzyme and substrates and products aids in the elucidation of catalytic pathways.;The manganese superoxide dismutases (MnSOD) neutralize naturally occurring toxic superoxide radicals. Mutational analysis of human MnSOD demonstrated that Glu162, a second-shell ligand of the Mn ion is necessary for efficient activity, due to tuning of the Mn. Additionally, the eukaryotic MnSODs are typically more product-inhibited than their prokaryotic counterparts. Alteration of the active site mouth in human MnSOD resulted in a weakly product-inhibited form.;The carbonic anhydrases (CAs) are a family of structurally diverse enzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and a proton. Due to the high turnover rate, an understanding of enzyme-substrate interactions has been elusive. The use of a high-pressure environment allowed for successful capture of carbon dioxide in the hydrophobic pocket in the active site of human CA II. The x-ray crystal structure of both zinc-bound and zinc-free HCA II revealed that the active site remains relatively static, acting as a solvation site for CO2, thus allowing for rapid turnover.;A proton transfer step is also required in the catalytic cycle of CA to allow for the regeneration of the active zinc-bound hydroxide. This is accomplished by proton transfer along a solvent-mediated proton wire, leading to the final proton acceptor. Mutational analysis of the environment surrounding the proton shuttle residue, His64, in HCA II revealed that the enzyme finely tunes this region to allow for bidirectional proton transfer under physiological conditions. Mutational analysis of a gamma-CA revealed that residues located adjacent to the active site affect the proton transfer properties in this enzyme. These data suggest that one must carefully consider residues outside the active site environment when analyzing the catalytic activity of an enzyme.