Protein Mass-Modulated Effects in the Catalytic Mechanism of Dihydrofolate Reductase: Beyond Promoting Vibrations

Abstract

The role of fast protein dynamics in enzyme catalysis has been of great interest in the past decade. Recent “heavy enzyme” studies demonstrate that protein mass-modulated vibrations are linked to the energy barrier for the chemical step of catalyzed reactions. However, the role of fast dynamics in the overall catalytic mechanism of an enzyme has not been addressed. Protein mass-modulated effects in the catalytic mechanism of Escherichia coli dihydrofolate reductase (ecDHFR) are explored by isotopic substitution (13C, 15N, and non-exchangeable 2H) of the wild-type ecDHFR (l-DHFR) to generate a vibrationally perturbed “heavy ecDHFR” (h-DHFR). Steady-state, pre-steady-state, and ligand binding kinetics, intrinsic kinetic isotope effects (KIEint) on the chemical step, and thermal unfolding experiments of both l- and h-DHFR show that the altered protein mass affects the conformational ensembles and protein–ligand interactions, but does not affect the hydride transfer at physiological temperatures (25–45 °C). Below 25 °C, h-DHFR shows altered transition state (TS) structure and increased barrier-crossing probability of the chemical step compared with l-DHFR, indicating temperature-dependent protein vibrational coupling to the chemical step. Protein mass-modulated vibrations in ecDHFR are involved in TS interactions at cold temperatures and are linked to dynamic motions involved in ligand binding at physiological temperatures. Thus, mass effects can affect enzymatic catalysis beyond alterations in promoting vibrations linked to chemistry.

Chemical Ligation and Isotope Labeling to Locate Dynamic Effects during Catalysis by Dihydrofolate Reductase

“…The circular dichroism (CD) spectra of ligated EcDHFR, NT‐EcDHFR, and CT‐EcDHFR revealed only minor differences to the spectrum of the wild‐type enzyme, suggesting that the secondary structural organization of EcDHFR is not sensitive to the modifications made for expressed protein ligation or to isotope substitution (Figure S5), an observation that is in agreement with previous CD analyses of completely isotopically substituted EcDHFR 1f,g. However, thermal unfolding and binding kinetic investigations had shown that heavy isotope substitution can alter the ground‐state conformational ensemble and ligand interactions of EcDHFR 1f.…”

“…However, thermal unfolding and binding kinetic investigations had shown that heavy isotope substitution can alter the ground‐state conformational ensemble and ligand interactions of EcDHFR 1f. Michaelis constants ( K M ) of ligated EcDHFR for NADPH and H 2 F were 7.6±2.4 and 0.4±0.1 μ m (Table S2) at pH 7, similar to those of the wild‐type enzyme (4.8±1.0 and 0.7±0.2 μ m , respectively) 10.…”

Isotope-specific and amino acid-specific heavy atom substitutions alter barrier crossing in human purine nucleoside phosphorylase

“…The role of fast (femtosecond) enzyme dynamics in the catalytic cycle has remained elusive and controversial due to the experimental challenge of probing femtosecond motions in enzymes. Previous studies from our laboratory and others have reported that isotopic substitution to create heavy enzymes ( 13 C, 15 N, and 2 H) often slows catalytic site chemistry and, in some cases, alters rate constants for nonchemical steps (9)(10)(11)(12). Reduced catalytic site barrier-crossing (the chemical step) in isotopically labeled enzymes supports coupling of local femtosecond motion to transition-state formation and, in some cases, interaction with slower modes (10,11).…”

“…Fully labeled [ 2 H, 13 C, 15 N]PNP showed a 1.36 enzyme KIE for the chemical step of guanosine phosphorolysis (9). Although attributed to altered bond frequency in the enzyme-reactant complex to slow barrier-crossing, enzyme isotope effects with deuterium labeling have been questioned because of the possibility that physical differences between carbon-hydrogen and carbon-deuterium may alter protein architecture (11,19). We tested that hypothesis by separating the deuterium effects from the heavy-atom 13 13 C, 15 N]PNP.…”

Quantum Mechanics/Molecular Mechanics modeling of biological relevant reactions catalyzed by enzymes

“…A powerful tool to probe the nature of enzyme motions and their response to changes in the reaction conditions is the study of "enzyme isotope effects". [43][44][45][46][47][48][49][50][51][52][53][54][55] The coupling of protein motions to enzyme catalysis is revealed as a difference between the kinetic properties of the isotopologous enzymes, because mass-dependent translational, vibrational and rotational motions are altered by heavy isotope substitution, whereas the potential energy surface and electrostatic properties are unaffected. [45,46] -Covalent catalysis [4] : It suggests that the increase in the reaction rate is due to the temporary formation of a covalent bond between the enzyme and the substrate.…”