Mass-Dependent Bond Vibrational Dynamics Influence Catalysis by HIV-1 Protease

Self Cite

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...

mentioning

confidence: 70%

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

Suárez

Schramm

2015

Self Cite

“…Isotopic substitution of HIV protease, purine nucleoside phosphorylase, alanine racemase, and pentaerythritol tetranitrate reductase has been proposed to affect catalysis by changing ultrafast vibrations that couple to the reaction coordinate (44)(45)(46)(47). However, the precise manner in which such mass-dependent effects impact the different terms of the preexponential factor in Eq.…”

Section: Significancementioning

confidence: 99%

Unraveling the role of protein dynamics in dihydrofolate reductase catalysis

Luk

Ruiz‐Pernía

Dawson

et al. 2013

126

262

Protein dynamics have controversially been proposed to be at the heart of enzyme catalysis, but identification and analysis of dynamical effects in enzyme-catalyzed reactions have proved very challenging. Here, we tackle this question by comparing an enzyme with its heavy ( 15 N, 13 C, 2 H substituted) counterpart, providing a subtle probe of dynamics. The crucial hydride transfer step of the reaction (the chemical step) occurs more slowly in the heavy enzyme. A combination of experimental results, quantum mechanics/molecular mechanics simulations, and theoretical analyses identify the origins of the observed differences in reactivity. The generally slightly slower reaction in the heavy enzyme reflects differences in environmental coupling to the hydride transfer step. Importantly, the barrier and contribution of quantum tunneling are not affected, indicating no significant role for "promoting motions" in driving tunneling or modulating the barrier. The chemical step is slower in the heavy enzyme because protein motions coupled to the reaction coordinate are slower. The fact that the heavy enzyme is only slightly less active than its light counterpart shows that protein dynamics have a small, but measurable, effect on the chemical reaction rate.kinetics | computational chemistry | biological chemistry | biophysics | quantum biology

“…Bell-shaped pH-rate profiles (8)(9)(10)(11)(12) have revealed an acidic pH optimum, and structural studies (13,14) suggest that the reactant-bound enzyme contains a single protonated Asp (with a di-Asp, net charge of −1) facilitating a general acid-base mechanism, which is common among many Asp proteases (7,15). Nucleophilic attack by the water generates a reversible gem-diol intermediate, (3) which has been observed structurally (16)(17)(18) and identified experimentally (19,20). A series of kinetic studies have concluded that the breakdown of 3 to generate the products (7) is the rate-liming chemical step (9-11, 19, 21), denoted by k 5 .…”

mentioning

confidence: 99%

Transition states of native and drug-resistant HIV-1 protease are the same

Kipp¹,

Hirschi²,

Wakata³

et al. 2012

Self Cite

HIV-1 protease is an important target for the treatment of HIV/ AIDS. However, drug resistance is a persistent problem and new inhibitors are needed. An approach toward understanding enzyme chemistry, the basis of drug resistance, and the design of powerful inhibitors is to establish the structure of enzymatic transition states. Enzymatic transition structures can be established by matching experimental kinetic isotope effects (KIEs) with theoretical predictions. However, the HIV-1 protease transition state has not been previously resolved using these methods. We have measured primary 14 at the carbonyl carbon, proton transfer to the amide nitrogen leaving group, and C-N bond cleavage. A transition structure consistent with the KIE values involves proton transfer from the active site Asp-125 (1.32 Å) with partial hydrogen bond formation to the accepting nitrogen (1.20 Å) and partial bond loss from the carbonyl carbon to the amide leaving group (1.52 Å). The KIEs measured for the native and I84V enzyme indicate nearly identical transition states, implying that a true transition-state analogue should be effective against both enzymes.aspartyl protease | protease mechanism | transition-state structure | drug design