A fundamental problem in biochemistry is to understand how enzymes work. This project seeks greater insight into how nature designs enzyme active sites, particularly the residues that are not in direct contact with the reacting substrate molecule(s). Structural and biochemical studies have now characterized the active sites of hundreds of enzymes, and nearly all of these studies have focused on the amino acids in direct contact with the reacting substrate; these residues may be regarded as the first layer of the active site. Recent theoretical predictions and experimental studies strongly suggest that residues outside the first layer of the active site can also be very important for catalysis. The goals of this project are to combine theory, computation, and experiment to establish the prevalence of spatially extended active sites in enzymes, to show that this phenomenon is predictable computationally, and to elucidate the mechanisms by which spatially remote residues participate in enzyme catalysis. One such mechanism, and a problem of intense current interest, is the role of protein dynamics in the catalytic process. The examples studied here have been chosen because they have different kinds of interesting dynamical processes in play. In DNA polymerase III, motions in the polymerase subunit modulate DNA binding and are likely responsible for checking the fidelity of base-pair formation. For ornithine transcarbamylase, an induced fit conformational change has been reported to occur with the binding of the first reactant molecule. For glycinamide ribonucleotide transformylase, a pH-dependent molecular switch has been proposed for a coil-to-helix transition that activates the catalytic site. Preliminary evidence suggests that residues outside the first shell play central roles in each of these processes. For these three enzymes, mutations will be made at positions in the second and third shells that are predicted computationally to be important for catalysis, and these mutants will be characterized kinetically and structurally. Molecular dynamics (MD) simulations will be performed on the wild type and variants to determine whether the mutations affect the dynamics of the protein, and whether this motion contributes to catalysis, predictions that will be tested experimentally using wide-angle x-ray solution (WAXS) scattering. The establishment of principles governing remote residue participation in enzyme catalysis, and evidence that such participation is predictable computationally, will be very useful for enzyme engineering and mechanistic studies.
The computational methods developed for this project will be made freely available to the scientific community via the web for use in research, including protein engineering and enzyme mechanism studies, and in commercial applications. This project will provide better understanding of nature's design of enzyme active sites and of how enzymes affect catalysis. Such improved understanding can help in the development of novel technologies, such as cleaner, "green" industrial processes, sustainable methods for environmental remediation, and enzymatic biofuel synthesis. The training of highly qualified scientists as described in this project is vital to the regional high-tech economy and to U.S. competitiveness in the global economy. Two doctoral students will be trained in computational methods, in protein expression, mutation, purification, kinetics and binding assays, crystal structure determination, and other x-ray scattering methods. Parts of the project will be integrated into the Molecular Modeling course, with active student participation in some of the computational and modeling work. Research participation by undergraduate students will continue. This project will continue current collaborations with faculty members at primarily undergraduate institutions (PUIs), including a Hispanic-serving PUI. Hands-on demonstrations of computational and other research tools will continue at PUIs and to inner city K-12 students. Extensive participation by project team members in Native American student, professional, and community groups will continue.
