Dynamical activation of function in metalloenzymes

Abstract

Formulations of hydrogen tunneling in enzyme‐catalysed C–H activation reactions indicate enthalpic barriers to reaction that are independent of chemical steps and dependent on the protein scaffold. A tool to identify catalytically relevant site‐specific protein thermal networks has emerged from temperature‐dependent hydrogen deuterium exchange (TDHDX). Focusing on mutant enzyme forms with altered activation energies for catalysis, TDHDX provides a comparative analysis of the impact of mutation on Ea for local protein unfolding. Identified thermal networks appear unrelated to protein scaffold conservation and track to the dictates of the catalysed reaction, including sites for metal binding. The positions of thermal networks provide a framework for further understanding of time‐dependent, functionally relevant protein motions. Measurement of nanosecond Stokes shifts at the surface of the thermal network in soybean lipoxygenase yields activation energies that are identical to Ea values measured for kcat. This finding identifies a rapid (> nanosecond), long‐range and cooperative structural reorganization as the thermal barrier to catalysis. A model for protein dynamics is put forward that integrates broadly distributed protein conformational sampling with protein embedded thermal networks.

Identification of the Thermal Activation Network in Human 15-Lipoxygenase-2: Divergence from Plant Orthologs and Its Relationship to Hydrogen Tunneling Activation Barriers

“…Mutation-induced perturbations of the enzyme activity can be accompanied by deviations in the protein flexibility as either more flexible or less flexible (i.e., 0 < ΔE a,HDX < 0). 5,28,30 From the analysis, six nonoverlapping peptides were identified with elevated E a,HDX values and are almost exclusively attributed to the V426A substitution (Figure 4A). The trends in the ΔE a , HDX values track with the observed increase in the activation energy for catalysis, associated with the V426A mutation (Δ ∼ + 5 kcal/mol; Table S3).…”

“…This restructuring is considered the source of the E a values for tunneling in SLO. 5,6 The SLO reaction rates are weakly temperature-dependent (E a = 2 kcal/mol). 49 The human 15-LOX-2 reaction with AA, however, has a larger activation energy of 7 kcal/mol (Tables S3 and S4) and is within the range reported for other animal LOXs.…”

“…24−26 The incorporation of variable temperature in HDX-MS analysis has recently extended the study to thermally activated changes in the flexibility of a protein, of which a subset of peptide fluctuations can then be linked to catalysis through comparative studies using site-selective, protein mutations with well-defined catalytic impairments. 5 Employing this technique in conjunction with volume-reducing mutations of bulky, aliphatic residues initially for SLO led to the identification of a remote, solvent-exposed loop, residues 317−334 (Figure 1A, red loop), that showed correlated activation energies for both the H/D exchange process and the catalytic tunneling process. 15 Subsequent time-resolved spectroscopic and room-temperature X-ray structural studies revealed that a long-range, anisotropic structural reorganization of the protein, involving the identified solvent loop, is the origin of the enthalpic barrier (i.e., E a ) for hydrogen tunneling in SLO.…”

“…Native enzymes perform with unparalleled catalytic power, achieving reaction-rate enhancement capabilities exceeding 20 orders of magnitude. , In addition to the recognition of conformational changes needed for substrate binding and product release, there is increasing evidence for the role of protein motions in modulating the barrier height and width to attain reaction rates in a biologically viable range. − Several studies have identified energetically coupled networks in proteins that have been implicated in energy transfer from the solvent–protein interface to buried active sites to mediate thermally activated protein function. − Because protein structures fold with nonuniform packing density, thermal energy transport is expected to be anisotropic . The role of protein dynamics as it relates to catalysis is particularly evident in enzymes that mediate homolytic C–H bond cleavage, in which the net formal hydrogen atom (H·) is transferred quantum mechanically. − In these reactions, there is an innate, although not well understood, relationship between the classical properties of the protein (i.e., protein dynamics or conformational fluctuations) and the ability to enhance quantum features under the “noise” of biologically accessible conditions.…”

Nobel symposium #168 Visions of bio‐inorganic chemistry: metals and the molecules of life

“…The article describes the current state of knowledge as well as how the recent characterization of the ternary intermediate of Tyrosinase has contributed to the longstanding goal of understanding the monooxygenation mechanism of binuclear copper monooxygenases. Klinman [9] paints a fascinating picture of how function in metalloenzymes can be activated by dynamics and how the interplay between protein dynamics and function can be addressed using novel experimental and theoretical approaches. Broderick and Hoffman review the vast "radical SAM" family of proteins [10].…”