ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTEnzyme Catalysis: Beyond Classical Paradigms†Amnon Kohen and Judith P. KlinmanView Author Information Departments of Chemistry Molecular Cell Biology, University California at Berkeley, 94720 Cite this: Acc. Chem. Res. 1998, 31, 7, 397–404Publication Date (Web):May 30, 1998Publication History Received13 August 1997Published online30 May 1998Published inissue 1 July...

H-transfer was studied in the complex kinetic cascade of dihydrofolate reductase. Intrinsic isotope effects, their temperature dependence, and other temperature-dependent parameters indicated H-tunneling, but no 1° to 2° coupled motion. The data also suggested environmentally tunneling commitment catalysis on pre-steady-state effects.

One of the most intriguing questions in modern enzymology is whether enzyme dynamics evolved to enhance catalyzed chemical transformation. In this study, dihydrofolate reductase, a small monomeric protein that catalyzes single C-H-C transfer, used as model system address question. Experimental and computational studies have proposed dynamic network includes two residues remote from active site (G121 M42). The current study compares nature H-transfer step WT enzyme, mutants, their double...

Ultrafast two-dimensional infrared (2D IR) spectroscopy probes femtosecond to picosecond time scale dynamics ranging from solvation protein motions. The frequency-frequency correlation function (FFCF) is the quantitative measure of spectral diffusion that reports those and, within certain approximations, can be extracted directly 2D IR line shapes. A variety methods have been developed extract FFCF spectra, which, in principle, should give same parameters, but complexity real experimental...

This paper draws attention to selected experiments on enzyme-catalyzed reactions that show convex Arrhenius plots, which are very rare, and points out Tolman's interpretation of the activation energy places a fundamental model-independent constraint any detailed explanation these reactions. The analysis presented here shows in such systems, rate coefficient as function is not just increasing more slowly than expected, it actually decreasing. data provides proposed microscopic models, i.e.,...

In this paper, the Arrhenius curves of selected hydrogen-transfer reactions for which kinetic data are available in a large temperature range reviewed. The discussed terms one-dimensional Bell–Limbach tunnelling model. main parameters model barrier heights isotopic reactions, width H-reaction, masses, pre-exponential factor and minimum energy to occur. allows one compare different simple way prepare more-dimensional treatments. first type is concerned with where geometries reacting molecules...

A significant contemporary question in enzymology involves the role of protein dynamics and hydrogen tunneling enhancing enzyme catalyzed reactions. Here, we report a correlation between donor–acceptor distance (DAD) distribution intrinsic kinetic isotope effects (KIEs) for dihydrofolate reductase (DHFR) reaction. This study compares nature hydride-transfer step series active-site mutants, where size side chain that modulates DAD (I14 E. coli DHFR) is systematically reduced (I14V, I14A,...

The structure of formate dehydrogenase from Candida boidinii (CbFDH) is both academic and practical interests. First, this enzyme represents a unique model system for studies on the role protein dynamics in catalysis, but so far these have been limited by availability structural information. Second, CbFDH its mutants can be used various industrial applications (e.g., CO2 fixation or nicotinamide recycling systems), lack information has limiting factor commercial development. Here, we report...

The role of femtosecond-picosecond structural dynamics proteins in enzyme-catalyzed reactions is a hotly debated topic. We report infrared photon echo measurement the formate dehydrogenase-NAD+-azide ternary complex. In contrast to earlier studies protein dynamics, data show complete spectral diffusion on time scale with no static heterogeneity. This result indicates that this transition-state analogue complex completely samples distribution structures determine azide vibrational frequencies...

For several decades the hydride transfer catalyzed by alcohol dehydrogenase has been difficult to understand. Here we add large corpus of anomalous and paradoxical data collected for this reaction measuring a normal (> 1) 2 degrees kinetic isotope effect (KIE) reduction benzaldehyde. Because relevant equilibrium is inverse (< 1), KIE eludes traditional interpretation KIEs. It does, however, enable development comprehensive model "tunneling ready state" (TRS) that fits into general scheme...

The potential for femtosecond to picosecond time-scale motions influence the rate of intrinsic chemical step in enzyme-catalyzed reactions is a source significant controversy. Among central challenges resolving this controversy difficulty experimentally characterizing thermally activated at time scale functionally relevant enzyme complexes. We report series measurements address problem using two-dimensional infrared spectroscopy characterize scales active-site complexes formate dehydrogenase...

In recent years, the temperature dependence of primary kinetic isotope effects (KIE) has been used as indicator for physical nature enzyme-catalyzed H-transfer reactions. An interactive study where experimental data and calculations examine same chemical transformation is a critical means to interpret more properly KIEs. Here, rate-limiting step thymidylate synthase-catalyzed reaction studied by hybrid quantum mechanics/molecular mechanics (QM/MM) simulations in theoretical framework...

The role of fast protein dynamics in enzyme catalysis has been great interest the past decade. Recent "heavy enzyme" studies demonstrate that mass-modulated vibrations are linked to energy barrier for chemical step catalyzed reactions. However, overall catalytic mechanism an not addressed. Protein effects Escherichia coli dihydrofolate reductase (ecDHFR) explored by isotopic substitution (13C, 15N, and non-exchangeable 2H) wild-type ecDHFR (l-DHFR) generate a vibrationally perturbed ecDHFR"...

Significance Dihydrofolate reductase is a classic drug target because it promotes the NADPH-dependent reduction of 7,8-dihydrofolate (DHF) to yield 5,6,7,8-tetrahydrofolate (THF), which involved in biosynthesis purines, thymidylate, and several amino acids. It also popular model system for various biochemical/biophysical studies. However, there are many unresolved mechanistic issues regarding mechanism catalysis. We combined primary, solvent, multiple kinetic isotope effects; their...

Kinetic isotope effects (KIEs) and their temperature dependence can probe the structural dynamic nature of enzyme-catalyzed proton or hydride transfers. The molecular interpretation requires expensive specialized quantum mechanics/molecular mechanics (QM/MM) calculations to provide a quantitative understanding. Currently available phenomenological models use nonadiabatic assumption that is not appropriate for most proton-transfer reactions, while others require more parameters than...

Kinetic Isotope effects (KIEs) have long served as a probe for the mechanisms of both enzymatic and solution reactions. Here, we discuss various models physical sources KIEs, how experimentalists can use those to interpret their data, focus traditional has grown model that includes motion enzyme quantum mechanical nuclear tunneling. We then present two case studies enzymes, thymidylate synthase alcohol dehydrogenase, KIEs shed light on C-H bond cleavages enzymes catalyze. will show...

Three glycoforms of glucose oxidase, which vary in their degree glycosylation and resulting molecular weight, have been characterized with regard to catalytic properties. Focusing on 2-deoxyglucose probe the chemical step, we now measured temperature dependence competitive H/T D/T kinetic isotope effects enthalpy activation using [1-2H]-2-deoxyglucose. The effect Arrhenius preexponential factor (AD/AT) is 1.47 (±0.09), 1.30 (±0.10), 0.89 (±0.04) for 136, 155, 205 kDa glycoforms,...

Since the early days of enzymology attempts have been made to deconvolute various contributions physical phenomena enzyme catalysis. Here we present experimental and theoretical studies that examine possible role hydrogen tunneling, coupled motion, dynamics in In this review, first introduce basic concepts catalysis from a chemistry point view. Then, several recent developments application tools can probe dynamic effects other may contribute These include kinetic isotope (KIEs), their...