Acceleration of catalysis in dihydrofolate reductase by transient, site-specific photothermal excitation

Abstract

Significance Direct excitation of specific protein structural dynamics as a test of their role in enzyme catalysis has not yet been possible. Here we show that such motions can be excited photothermally, using a gold nanoparticle attached to a specific site on the enzyme dihydrofolate reductase (DHFR). DHFR is known to adopt multiple ground-state conformations having different kinetics of enzyme turnover. Site-specific photothermal excitation enables the enzyme to sample different reactive basins in the catalytic energy landscape and accelerates catalysis. We postulate that transient, site-specific heating excites a network of coupled motions that form a collective breathing mode of the protein structure near the active site, facilitating redistribution of the population into more reactive conformations. We have studied the role of protein dynamics in chemical catalysis in the enzyme dihydrofolate reductase (DHFR), using a pump–probe method that employs pulsed-laser photothermal heating of a gold nanoparticle (AuNP) to directly excite a local region of the protein structure and transient absorbance to probe the effect on enzyme activity. Enzyme activity is accelerated by pulsed-laser excitation when the AuNP is attached close to a network of coupled motions in DHFR (on the FG loop, containing residues 116–132, or on a nearby alpha helix). No rate acceleration is observed when the AuNP is attached away from the network (distal mutant and His-tagged mutant) with pulsed excitation, or for any attachment site with continuous wave excitation. We interpret these results within an energy landscape model in which transient, site-specific addition of energy to the enzyme speeds up the search for reactive conformations by activating motions that facilitate this search.