David N Silverman, Ph.D.

Dr. Silverman’s lab studied the catalytic mechanism of two very fast enzymes, carbonic anhydrase and superoxide dismutase. They asked what accounts for the extraordinary efficiency of these enzymes, which are able to convert the substrates carbon dioxide and superoxide to products with catalytic turnovers as great as one million per second. Both enzymes are involved in critical physiological processes. The carbonic anhydrases comprise many isoforms in humans that convert carbon dioxide into bicarbonate to form secretory fluids and in the red cells’ function during respiration. The superoxide dismutases convert superoxide, a toxic by-product of many enzymatic reactions, into hydrogen peroxide and oxygen.

With carbonic anhydrase, their work focused mainly on the mechanism of proton transfer in the active site. In the pathway, protons must be transferred from the zinc-bound water to solution at a rate at least as fast as a million per second. There is a proton shuttle residue, His64 in many carbonic anhydrase isozymes, that accepts a proton from the zinc-bound water through intervening hydrogen-bonded water bridges and transfers it to the solution. They measured the rates of intramolecular proton transfer in many isozymes and site-specific mutants of carbonic anhydrase to understand what processes determine the activation barrier for this proton transfer rate. Among the possibilities are the energy to orient side chains, such as His64, and the energy to form a hydrogen-bonded water chain between the zinc and His64. This is a very good system for studying such proton transfers, and the properties of intramolecular proton transfer that they discovered should be applicable to more complex systems such as bacteriorhodopsin, cytochrome c oxidase, and F0F1 ATPase, among others.

Their work with manganese superoxide dismutase was, in some ways, similar to that on carbonic anhydrase to determine the pathway and rates of the proton transfers that form the product hydrogen peroxide. This enzyme has a network of hydrogen-bonded water and side chains that extend throughout the active site. Their work with site-specific mutants determined that this network plays a critical role in these proton transfers. They also studied the role of nearby residues on the redox potential of the manganese and how this influences the catalysis. Under many conditions, manganese superoxide dismutase is limited in rate by a product inhibition. Using a variety of methods, they measured the rates of formation of the inhibited complex and identified its structure.

Much of their work involved measuring the rates of very fast enzymatic reactions. They used several methods in their work on carbonic anhydrase and superoxide dismutase. Stopped-flow spectrophotometry is capable of measuring the optical properties of solutions containing enzyme and substrate after a mixing time of about 1.5 milliseconds. Their instrumentation could measure a full absorption spectrum about every millisecond after that. With superoxide dismutase, reactions are often faster than that, in which case they used pulse radiolysis which relies on an electron accelerator at the Brookhaven National Laboratory. They also found that mass spectrometry to measure the enzyme catalyzed exchange of oxygen-18 between carbon dioxide and water is very informative for carbonic anhydrase. Through collaborations, they had projects to determine structure using x-ray diffraction and to determine the properties of the manganese using electron paramagnetic resonance, as well as studies of stabilities using scanning differential calorimetry.