Enzymes are highly dynamic molecules, but the role of protein dynamics in their mechanism of chemical catalysis is not well understood. A direct approach to test the role of specific protein structural dynamics in enzyme catalysis would be to measure the effect on catalysis of excitation of such motions. We have studied the role of protein dynamics in the enzyme dihydrofolate reductase (DHFR), by photothermal excitation of local structural dynamics and transient absorbance spectroscopy to probe the effect on enzyme activity. Pulsed-laser photothermal heating of a gold nanoparticle (AuNP) attached site-specifically to DHFR directly excites a local region of the protein structure. When the AuNP is attached close to a network of coupled motions in DHFR (on the FG loop or on a nearby alpha helix), enzyme activity is accelerated by pulsed laser excitation. Conversely, no rate acceleration is observed when the AuNP is attached at a site that is distant from the network (Distal Mutant and His-tagged Mutant) with pulsed excitation, or for any attachment site with continuous wave excitation. We have used an energy landscape model to interpret these results. DHFR adopts multiple ground state conformations, each having different kinetics of enzyme turnover. Transient, site-specific addition of energy to the network of coupled motions speeds up the search for reactive conformations by enabling the enzyme to sample different reactive basins in the catalytic energy landscape. Excitation of the network of coupled motions, which form a collective breathing mode of the protein structure near the active site, facilitates the redistribution of the population into more reactive conformations.

Photothermal excitation of a network of coupled motions in dihydrofolate reductase accelerates catalysis

ACS Fall 2021

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