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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM025765-37
Application #
8653571
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Barski, Oleg
Project Start
1978-06-01
Project End
2016-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
Support Year
37
Fiscal Year
2014
Total Cost
$518,017
Indirect Cost
$187,016
Name
University of California Berkeley
Department
Miscellaneous
Type
Organized Research Units
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Klinman, Judith P; Kohen, Amnon (2014) Evolutionary aspects of enzyme dynamics. J Biol Chem 289:30205-12
Carr, Cody A Marcus; Klinman, Judith P (2014) Hydrogen tunneling in a prokaryotic lipoxygenase. Biochemistry 53:2212-4
Hu, Shenshen; Sharma, Sudhir C; Scouras, Alexander D et al. (2014) Extremely elevated room-temperature kinetic isotope effects quantify the critical role of barrier width in enzymatic C-H activation. J Am Chem Soc 136:8157-60
Klinman, Judith P (2014) The power of integrating kinetic isotope effects into the formalism of the Michaelis-Menten equation. FEBS J 281:489-97
Meadows, Corey W; Tsang, Jonathan E; Klinman, Judith P (2014) Picosecond-resolved fluorescence studies of substrate and cofactor-binding domain mutants in a thermophilic alcohol dehydrogenase uncover an extended network of communication. J Am Chem Soc 136:14821-33
Meadows, Corey W; Ou, Ryan; Klinman, Judith P (2014) Picosecond-resolved fluorescent probes at functionally distinct tryptophans within a thermophilic alcohol dehydrogenase: relationship of temperature-dependent changes in fluorescence to catalysis. J Phys Chem B 118:6049-61
Klinman, Judith P; Kohen, Amnon (2013) Hydrogen tunneling links protein dynamics to enzyme catalysis. Annu Rev Biochem 82:471-96
Osborne, Robert L; Zhu, Hui; Iavarone, Anthony T et al. (2013) Interdomain long-range electron transfer becomes rate-limiting in the Y216A variant of tyramine ýý-monooxygenase. Biochemistry 52:1179-91
Johnson, Bryan J; Yukl, Erik T; Klema, Valerie J et al. (2013) Structural snapshots from the oxidative half-reaction of a copper amine oxidase: implications for O2 activation. J Biol Chem 288:28409-17
Nagel, Zachary D; Cun, Shujian; Klinman, Judith P (2013) Identification of a long-range protein network that modulates active site dynamics in extremophilic alcohol dehydrogenases. J Biol Chem 288:14087-97

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