Understanding how enzymes work has constituted the core of basic research in medically related fields for over 50 years. Such studies have led to the description of a vast array of different enzyme classes that includes their 3-dimensional structures and underlying chemical mechanisms. For most of this time, the fundamental assumption regarding the origin of enzyme catalysis has remained wedded to a mid-20th century proposal referred to as """"""""enhanced transition state binding"""""""". For the last decade, this monolithic and static description of catalysis has given way to the challenging question regarding the role of protein size and motions in achieving huge rate accelerations. While the inherent flexibility of proteins had long been recognized as important to function, for example in models of allostery, the direct link of protein motions to the chemistry at enzyme active sites has been difficult to access experimentally. This proposal describes a series of experimental approaches aimed at tackling this challenging and central problem regarding enzyme function. The systems chosen for further characterization catalyze fundamental and pervasive processes in biology (hydride and hydrogen atom transfer reactions). Studies of this nature are central to the development of robust models for biological catalysis that can guide future efforts at drug design and de novo protein design. There are three main goals for this proposal. First, a family of temperature-adapted, tetrameric prokaryotic alcohol dehydrogenases (ADHs) has been described that includes highly homologous thermophilic (ht- ADH) and psychrophilic (ps-ADH) variants. Completed kinetic characterizations implicate hydride transfer via hydrogenic wave function overlap between donor and acceptor atoms that is linked to local hydrophobic side chains and conformational landscapes. The specific protein motions controlling H-tunneling will be studied using a combination of fluorescence lifetime and anisotropy measurements, resonance Raman spectroscopy and hydrogen deuterium exchange. A link of active site flexibility to a remote specific side chain at the protein dimer interface has been identified and will be tested using kinetic, spectroscopic and protein chemistry probes. Second, the role of protein motions in catalysis will be pursued for a paradigmatic hydrogen atom tunneling reaction, lipoxygenase, where experimental approaches will include paramagnetic NMR, high-pressure kinetic studies and room temperature X-ray characterization. Third, detailed studies of tyramine-b-monooxygenase, a model for the mammalian, mononuclear/two- copper enzymes (dopamine b-monooxygenase and peptidylglycine-a-monooxygenase) will be focused on the inter-domain communication that controls hydrogen abstraction at CuM and long-range electron transfer from CuH to CuM.
During the last decade, the dominant focus on enhanced transition state binding as the origin of enzyme catalysis has given way to the challenging question of the role of protein motions and active site compression in achieving huge rate accelerations. This proposal describes a series of experimental approaches aimed at tackling this central problem regarding enzyme function. Studies of this nature are of importance to the development of robust models for biological catalysis that can guide future efforts at drug design and de novo protein design.
Showing the most recent 10 out of 92 publications
