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

Abstract

Computational chemistry predicts that atomic motions on the femtosecond timescale are coupled to transition-state formation (barrier-crossing) in human purine nucleoside phosphorylase (PNP). The prediction is experimentally supported by slowed catalytic site chemistry in isotopically labeled PNP ( 13 C, 15 N, and 2 H). However, other explanations are possible, including altered volume or bond polarization from carbon-deuterium bonds or propagation of the femtosecond bond motions into slower (nanoseconds to milliseconds) motions of the larger protein architecture to alter catalytic site chemistry. We address these possibilities by analysis of chemistry rates in isotope-specific labeled PNPs. Catalytic site chemistry was slowed for both heavy enzymes | transition state coupling | femtosecond dynamics | pre-steady-state chemistry | Born-Oppenheimer enzymes P rotein and atomic motions required for enzymatic catalysis involve a broad range of timescales from the millisecond to the femtosecond. Slower (millisecond) motions are involved in conformational changes that occur during substrate binding and product release (1-4), whereas femtosecond motions (promoting vibrations) are proposed by computational studies to be involved in the chemical step of transition-state formation (covalent bond changes) (5-8). 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 (9-12). 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).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. Transition path sampling with heavy and light PNPs studied reactive trajectories and predicted that femtosecond vibrational motions are less coupled in the heavy enzyme (14).PNP (EC 2.4.2.1) catalyzes the reversible phosphorolysis of 6-oxypurine (and 2′-deoxy)-β-D-ribonucleosides to yield (2-deoxy)-α-D-ribose 1-phosphate and the purine base (15) (Fig. 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 ch...

Untangling Heavy Protein and Cofactor Isotope Effects on Enzyme-Catalyzed Hydride Transfer

“…The 131 Da mass difference is consistent with loss of the N-terminal Met in vivo. Deuterated and triple-labeled enzymes have measured m/z = 42,618.9 and 44,627.1 Da respectively, which are 5.3 % and 10.3 % heavier, respectively, than the unlabeled enzyme, similar to other reports of deuterated 13 and triple-labeled e.g. 11,12,17,19 enzymes.…”

“…Like our previous report, 15 the coenzyme KIEs are more temperature dependent (larger ∆∆HT0 ‡ ) in heavy PETNR, but the contributions from the protein and FMN cofactor do not appear to be additive, with the mixed-label EhFl and ElFh isotopologues showing larger ∆∆HT0 ‡ values than EhFh PETNR. Together, these data show that both the protein and FMN contribute to the 'heavy enzyme' effect in PETNR, and suggest that the nature of the isotope ( 2 H vs. 13 C vs. 15 N) and the location of the isotopic substitution can perturb the reaction in different ways such that these effects may (partially) cancel each other out. The isotope effect spe- Direct experimental observation of the timescale(s) of any vibrations that couple the protein and/or FMN to the chemical coordinate is necessary in order to firmly establish the theoretical origin of the 'heavy enzyme' effect in PETNR.…”

“…[8][9][10] Recently, Schramm and colleagues showed that stable isotope-( 2 H, 13 C and/or 15 N) labeled 'heavy' purine nucleoside phosphorylase and HIV-1 protease enzymes have measurably slower reaction kinetics. [11][12][13][14] These data were initially interpreted in terms of the Born-Oppenheimer approximation, where increased protein mass (due to isotopically labeling) alters bond vibrational frequencies without affecting electrostatic properties of the enzyme (ionizable protons were not labeled). The authors suggested that the lower frequency of (fs) bond vibrations in the 'heavy enzymes' may lead to a reduction in conformational sampling and thus chemical barrier crossing, which is proportional to the rate of barrier crossing.…”

Catalytic-site design for inverse heavy-enzyme isotope effects in human purine nucleoside phosphorylase

“…The millisecond motions are linked to structural changes during substrate binding (1) and product release, whereas the femtosecond motions are involved in transition state (TS) formation. Alterations of the femtosecond dynamics by isotope substitution in enzymes influence the probability of TS barrier crossing when protein femtosecond motions are coupled to chemistry at catalytic sites (2,3).…”

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.…”