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

Abstract

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.

Temporal and Spatial Resolution of a Protein Quake that Activates Hydrogen Tunneling in Soybean Lipoxygenase

“…S1). The majority of the I552A-derived peptides display nearly identical exchange behavior to WT across the 5 temperatures (10,20,25,30, and 40 °C) analyzed (cf. Data S1).…”

“…S1). The majority of the I552Aderived peptides display nearly identical exchange behavior to WT across the 5 temperatures (10,20,25,30, and 40 °C) analyzed (SI Appendix, Data S1). The peptide of particular interest is 317-334 that represents the surface loop that was previously shown (19) to undergo correlated alterations in the activation energy for HDX Ea(kHDX) and Ea(kcat) when interrogating the variants I553G and L546A in relation to WT.…”

“…Temperature dependent hydrogen-deuterium exchange coupled to mass spectrometry (TDHDX-MS) has recently emerged as a broadly accessible experimental tool to interrogate heat induced changes in local protein flexibility, which when coupled to function-altering protein mutations provides spatial resolution of activated networks that are found to lead from protein-solvent interfaces to enzyme active sites (18)(19)(20)(21)(22). While the thermal activation of enzymes can be rationalized as a general redistribution of equilibrated protein states toward increasingly active configurations with increasing temperature, the growing identification of reaction specific thermal networks that are distinctive from each other and independent of protein scaffold conservation points toward scaffold-embedded heat conduits as a source of dynamical excitation (23)(24)(25)(26)(27)(28)(29)(30).…”

Protein Dynamics and Enzymatic Catalysis

“…There will be further discussion of this issue later in this Perspective when we discuss laboratory evolved artificial enzymes. While predicted over 11 years ago, this kind of energy transfer from the surface of the protein to the active site has now been observed experimentally in soybean lipoxygenase and also bacterial DHFR for slower motions …”

Single-Molecule Sampling of Dihydrofolate Reductase Shows Kinetic Pauses and an Endosteric Effect Linked to Catalysis

“…Dihydrofolate reductase (DHFR) has been used extensively as a model enzyme for investigating the link between structure, dynamics, and catalysis. − The enzyme catalyzes the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF) using nicotinamide adenine dinucleotide phosphate (NADPH) as the cofactor (Figure A). Five intermediates have been identified in the steady-state catalytic cycle and characterized by X-ray crystallography − and NMR: ,,,, E:NADPH, E:NADPH:DHF, E:NADP + :THF, E:THF, and E:NADPH:THF (Figure B).…”