Femtosecond dynamics coupled to chemical barrier crossing in a Born-Oppenheimer enzyme

Abstract

Contributions of fast (femtosecond) dynamic motion to barrier crossing at enzyme catalytic sites is in dispute. Human purine nucleoside phosphorylase (PNP) forms a ribocation-like transition state in the phosphorolysis of purine nucleosides and fast protein motions have been proposed to participate in barrier crossing. In the present study, 13 C−, 15 N−, 2 H−labeled human PNP (heavy PNP) was expressed, purified to homogeneity, and shown to exhibit a 9.9% increase in molecular mass relative to its unlabeled counterpart (light PNP). Kinetic isotope effects and steady-state kinetic parameters were indistinguishable for both enzymes, indicating that transition-state structure, equilibrium binding steps, and the rate of product release were not affected by increased protein mass. Single-turnover rate constants were slowed for heavy PNP, demonstrating reduced probability of chemical barrier crossing from enzyme-bound substrates to enzyme-bound products. In a second, independent method to probe barrier crossing, heavy PNP exhibited decreased forward commitment factors, also revealing mass-dependent decreased probability for barrier crossing. Increased atomic mass in human PNP alters bond vibrational modes on the femtosecond time scale and reduces on-enzyme chemical barrier crossing. This study demonstrates coupling of enzymatic bond vibrations on the femtosecond time scale to barrier crossing. femtosecond motions | kinetic isotope effect | transition state structure | enzymatic catalysis | protein dynamics

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

“…Several lines of evidence indicated that deuterium substitution does not alter the electrostatic properties of the active site and consequently the activation free energy,1a,b,g and that protein isotope labeling does not alter the tunneling coefficient despite the smaller size of a fully deuterated protein 1g,h,j. However, the recrossing trajectories were changed upon protein isotope labeling, thus giving a subtle difference in the hydride‐transfer rate constant:(2), 1g,h,j …”

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).…”

“…In early heavy-enzyme studies, Silva et al (9) uniformly labeled the amino acid residues of human purine nucleoside phosphorylase (PNP) with 13 C, 15 N, and nonexchangeable 2 H in an attempt to perturb local bond vibrational modes without altering the electrostatics of the enzyme (the Born-Oppenheimer approximation) (13). Heavy PNP demonstrated unchanged steady-state kinetic parameters, kinetic isotope effects, and transition state structure, but the chemical rate was decreased.…”

“…1). Isotopically labeled PNP expressed from 13 C and 15 N precursors in D 2 O solvent is 9.9% heavier than natural abundance PNP and shows a heavy enzyme kinetic isotope effect (KIE) of 1.36 (k chem light/k chem heavy) (9). Reduced catalytic site chemistry in heavy PNP was interpreted as slowed femtosecond atomic vibrational modes giving less-efficient coupling to transition-state formation (9,15) (Fig.…”