Network of coupled promoting motions in enzyme catalysis

A network of coupled promoting motions in the enzyme dihydrofolate reductase is identified and characterized. The present identification is based on genomic analysis for sequence conservation, kinetic measurements of multiple mutations, and mixed quantum/classical molecular dynamics simulations of hydride transfer. The motions in this network span time scales of femtoseconds to milliseconds and are found on the exterior of the enzyme as well as in the active site. This type of network has broad implications for an expanded role of the protein fold in catalysis as well as ancillaries such as the engineering of altered protein function and the action of drugs distal to the active site.     

Colossal kinetic isotope effects in proton-coupled electron transfer

The kinetics of reduction of benzoquinone (Q) to hydroquinone (H(2)Q) by the Os(IV) hydrazido (trans-[Os(IV)(tpy)(Cl)(2)(N(H)N(CH(2))(4)O)]-PF(6) = [1]PF(6), tpy = 2,2':6',2"-terpyridine), sulfilimido (trans-[Os(IV)-(tpy)(Cl)(2)(NS(H)-4-C(6)H(4)Me)]PF(6) = [2]PF(6)), and phosphoraniminato (trans-[Os(IV)(Tp)(Cl)(2)(NP(H)(Et)(2))] = [3], Tp(-) = tris(pyrazolyl)-borate) complexes have been studied in 1:1 (vol/vol) CH(3)CN/H(2)O and CH(3)CN/D(2)O (1.0 M in NH(4)PF(6)/KNO(3) at 25.0 +/- 0.1 degrees C). The reactions are first order in both [Q] and Os(IV) complex and occur by parallel pH-independent (k(1)) and pH-dependent (k(2)) pathways that can be separated by pH-dependent measurements. Saturation kinetics are observed for the acid-independent pathway, consistent with formation of a H-bonded intermediate (K(A)) followed by a redox step (k(red)). For the pH-independent pathway, k(1)(H(2)O)/k(1)(D(2)O) kinetic isotope effects are 455 +/- 8 for [1(+)], 198 +/- 6 for [2(+)], and 178 +/- 5 for [3]. These results provide an example of colossal kinetic isotope effects for proton-coupled electron transfer reactions involving nitrogen, sulfur, and phosphorus as proton-donor atoms.     

Anticorrelated motions as a driving force in enzyme catalysis: the dehydrogenase reaction

Molecular dynamics and cross-correlation analysis of the horse liver alcohol dehydrogenase HLADH.NAD(+).PhCH(2)O(-) complex has established anticorrelated motions between the NAD(+)-binding domain and other portions of the enzyme. Four pairs of anticorrelated interactions are (i and ii) cofactor-binding domain: C(alpha) of V292 and the CG1 of V203 with C7 of PhCH(2)O(-); (iii) cofactor-binding domain: amide carbonyl oxygen of I318 with amide N of H67; and (iv) cofactor domain: C(alpha) of T178 with carbonyl oxygen of L141. The average distances between pairs are 9.2 A for i, 8.2 A for ii, 14.7 A for iii, and 18.2 A for iv. The motions of i and ii are most important in the approximately 0.5 A pushing of C4 of NAD(+) toward C7 of PhCH(2)O(-) to form push near-attack conformer (NACs). The motions of iv are less so, and those of iii are not important. Seventy-five quantum mechanics/molecular mechanics calculations of the energies of reaction were carried out without structural restrictions from different stages of the molecular dynamics trajectory. Of the 71 conformations, the 29 fulfilling NAC criteria were associated with the lowest energies of activation. Thus, anticorrelated motions from the NAD(+)-binding domain by way of the neighboring V292 and V203 have a pushing motion, which moves the C4 of NAD(+) toward the H-C7 of the substrate. Longer-range anticorrelated motions involving the cofactor-binding domain have no or very little influence on NAC formation.     

An oxocarbenium-ion intermediate of a ribozyme reaction indicated by kinetic isotope effects

Many of the enzymes that catalyze reactions at nucleotide glycosidic linkages proceed through either a reactive oxocarbenium-ion intermediate or a transition state with considerable oxocarbenium character. To investigate how an RNA active site deals with the catalytic challenge of nucleotide synthesis, we probed the transition state of a ribozyme able to promote the formation of a pyrimidine nucleotide. Primary and secondary kinetic isotope effects indicate that this ribozyme stabilizes a highly dissociative reaction with considerable sp2 hybridization and negligible bond order between the departing pyrophosphate leaving group and the anomeric carbon. The small primary 13C isotope effect of 1.002 +/- 0.003 indicates that the reaction is likely to be less concerted than that observed for protein nucleotide synthesis enzymes, which typically have primary 13C isotope effects of 1.02-1.03. The dissociative nature of the ribozyme reaction most resembles the reaction of some hydrolytic enzymes, such as uracil DNA glycosylase, which uses the negative charges found in the phosphodiester backbone of its DNA substrate to transiently stabilize an oxocarbenium ion during hydrolysis. The detectable hydrolysis observed in the ribozyme reaction indicates that shielding of this reactive intermediate from water is a significant challenge for RNA, which protein enzymes that synthesize nucleotides have managed to overcome during evolution, apparently by the utilization of more concerted chemistry.     

Inverse heavy enzyme isotope effects in methylthioadenosine nucleosidases

Heavy enzyme isotope effects occur in proteins substituted with ²H-, ¹³C-, and ¹⁵N-enriched amino acids. Mass alterations perturb femtosecond protein motions and have been used to study the linkage between fast motions and transition-state barrier crossing. Heavy enzymes typically show slower rates for their chemical steps. Heavy bacterial methylthioadenosine nucleosidases (MTANs from Helicobactor pylori and Escherichia coli) gave normal isotope effects in steady-state kinetics, with slower rates for the heavy enzymes. However, both enzymes revealed rare inverse isotope effects on their chemical steps, with faster chemical steps in the heavy enzymes. Computational transition-path sampling studies of H. pylori and E. coli MTANs indicated closer enzyme–reactant interactions in the heavy MTANs at times near the transition state, resulting in an improved reaction coordinate geometry. Specific catalytic interactions more favorable for heavy MTANs include improved contacts to the catalytic water nucleophile and to the adenine leaving group. Heavy bacterial MTANs depart from other heavy enzymes as slowed vibrational modes from the heavy isotope substitution caused improved barrier-crossing efficiency. Improved sampling frequency and reactant coordinate distances are highlighted as key factors in MTAN transition-state stabilization.

Enzymes use slow (millisecond to second) motions to generate reactant-enzyme geometries near the transition state and are proposed to use fast catalytic site motions (femtosecond to picosecond) to find distinct transition-state conformations along the reaction coordinate. Chemical steps occur on the timescale of bond vibrations after the fast dynamic search achieves the transition-state conformation (1, 2). The isotopic labeling of enzymes with ¹³C, ¹⁵N, and nonexchangeable ²H is an experimental strategy for probing the contributions of fast protein motions in the crossing of transition-state barriers. These “heavy” enzymes have mass-altered vibrational modes but remain electrostatically unaltered from their natural counterparts due to principles of the Born-Oppenheimer approximation (1). The decrease in bond vibrational frequencies due to the increased molecular mass results in a slowed search for the transition state and, in most cases, a decreased rate for the chemical step (3, 4). Slower chemical steps have been observed in heavy enzyme studies of purine nucleoside phosphorylase (PNP), HIV-1 protease, alanine racemase, several dihydrofolate reductases (DHFR), pentaerythritol tetranitrate reductase (PETNR), lactate dehydrogenase, and in catalytic site mutations of some of these enzymes (4, 5).Inverse enzyme isotope effects are rare cases where the chemical reaction step of the heavy enzyme is faster than its light counterpart, only reported under conditions of engineered catalytic site mutations or specific catalytic site elements being isotopically labeled. In one case, the chemical step rates of heavy and light forms of a catalytic site mutant PNP (F159Y) yielded an inverse isotope effect (k_light/k_heavy for the chemical step) of 0.75 for the phosphorolysis of guanosine (6). In other examples, PNPs engineered with asparagines labeled differently from the rest of the protein gave inverse isotope effects of 0.71 and 0.78 (7). PETNR was reported to have an enzyme isotope effect of 0.89 for deuteride ion transfer involving NADP²H with labeled enzyme and the cofactor flavin mononucleotide (FMN) in its naturally abundant form. Another inverse enzyme isotope effect of 0.92 was reported for PETNR using NADP²H and heavy FMN (8). However, when PNP and PETNR were assayed in their fully labeled native and heavy forms with substrates containing a naturally abundant isotope composition, the reported enzyme kinetic isotope effects (KIEs) were normal (9). Here, we present two unusual cases of native enzymes with natural inverse isotope effects.Methylthioadenosine nucleosidases (MTANs) are dimeric microbial nucleoside hydrolases that catalyze the hydrolysis of the N-ribosidic bond of 5′-methylthioadenosine (MTA) to 5-methylthioribose and adenine. A broad specificity for substituents at the 5′-position of adenosine permits diverse metabolic roles in bacterial genera, including functions in purine salvage, quorum sensing, S-adenosylmethionine recycling, polyamine synthesis, and the futalosine-based menaquinone biosynthetic pathway. These roles of MTANs in bacterial metabolism have made them potential drug targets. For example, interfering with bacterial quorum sensing, but not growth, avoids the selective pressure of antibiotics, and prevents the development of resistance, while inhibiting the production of virulence factors (10). Blocking menaquinone synthesis in microorganisms using the futalosine-specific biosynthetic pathway selectively targets pathogenic bacteria, including Helicobacter and Campylobacter genera without affecting the broader gut microbiome (11–13).Transition-state structures of MTANs have been solved for various bacterial species by analysis of substrate KIEs. Early transition states, with significant bond order remaining to the adenine leaving group, have been assigned to the MTANs from Neisseria meningitidis and Helicobacter pylori. However, Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, and Klebsiella pneumoniae MTANs possess near-fully dissociated transition states (14). Mutational studies have highlighted the important roles that specific catalytic residues play in substrate binding and transition-state formation (15). Insight into the dynamic roles the catalytic site residues play in transition-state formation is a focus of this study.We selected MTANs from early (H. pylori MTAN; HpMTAN) and late (E. coli MTAN; EcMTAN) transition-state classes to determine the effects of heavy isotope-labeled protein. The enzymes showed normal heavy isotope effects on steady-state kinetic rates (k_cat values) and variable effects on substrate interactions (K_m values). But contrary to most enzyme systems, both MTANs revealed natural, intrinsic inverse isotope effects on the chemical step. Here we report the heavy enzyme MTAN isotope effects through kinetic experiments under steady-state and presteady-state conditions. Transition-path sampling is used to obtain mechanistic insight into how the coordination of dynamic motions at the catalytic sites confers this previously rare phenomenon. The discovery of natural inverse heavy enzyme isotope effects adds a new dimension to the usual observation that increased enzyme mass causes a decrease in the rate of the chemical step due to less-efficient barrier crossing. These findings also explain the mechanism of inverse effects and add to our understanding of rapid protein dynamic contributions to enzyme catalytic function.     

The kinetic basis of peptide exchange catalysis by HLA-DM

The mechanism by which the peptide exchange factor HLA-DM catalyzes peptide loading onto structurally homologous class II MHC proteins is an outstanding problem in antigen presentation. The peptide-loading reaction of class II MHC proteins is complex and includes conformational changes in both empty and peptide-bound forms in addition to a bimolecular binding step. By using a fluorescence energy transfer assay to follow the kinetics of peptide binding to the human class II MHC protein HLA-DR1, we find that HLA-DM catalyzes peptide exchange by facilitating a conformational change in the peptide-bound complex, and not by promoting the bimolecular MHC-peptide reaction or the conversion between peptide-receptive and -averse forms of the empty protein. Thus, HLA-DM serves essentially as a protein-folding or conformational catalyst.     

Characterizing semilocal motions in proteins by NMR relaxation studies

The understanding of protein function is incomplete without the study of protein dynamics. NMR spectroscopy is valuable for probing nanosecond and picosecond dynamics via relaxation studies. The use of ¹⁵N relaxation to study backbone dynamics has become virtually standard. Here, we propose to measure the relaxation of additional nuclei on each peptide plane allowing for the observation of anisotropic local motions. This allows the nature of local motions to be characterized in proteins. As an example, semilocal rotational motion was detected for part of a helix of the protein Escherichia coli flavodoxin.     

Dynamics and dissipation in enzyme catalysis

We use quantized molecular dynamics simulations to characterize the role of enzyme vibrations in facilitating dihydrofolate reductase hydride transfer. By sampling the full ensemble of reactive trajectories, we are able to quantify and distinguish between statistical and dynamical correlations in the enzyme motion. We demonstrate the existence of nonequilibrium dynamical coupling between protein residues and the hydride tunneling reaction, and we characterize the spatial and temporal extent of these dynamical effects. Unlike statistical correlations, which give rise to nanometer-scale coupling between distal protein residues and the intrinsic reaction, dynamical correlations vanish at distances beyond 4–6 Å from the transferring hydride. This work finds a minimal role for nonlocal vibrational dynamics in enzyme catalysis, and it supports a model in which nanometer-scale protein fluctuations statistically modulate—or gate—the barrier for the intrinsic reaction.     

Energetics of enzyme catalysis.

Quantitative studies of the energetics of enzymatic reactions and the corresponding reactions in aqueous solutions indicate that charge stabilization is the most important energy contribution in enzyme catalysis. Low electrostatic stabilization in aqueous solutions is shown to be consistent with surprisingly large electrostatic stabilization effects in active sites of enzymes. This is established quantitatively by comparing the relative stabilization of the transition states of the reaction of lysozyme and the corresponding reaction is aqueous solution.     

The mechanism of rate-limiting motions in enzyme function

The ability to use conformational flexibility is a hallmark of enzyme function. Here we show that protein motions and catalytic activity in a RNase are coupled and display identical solvent isotope effects. Solution NMR relaxation experiments identify a cluster of residues, some distant from the active site, that are integral to this motion. These studies implicate a single residue, histidine-48, as the key modulator in coupling protein motion with enzyme function. Mutation of H48 to alanine results in loss of protein motion in the isotope-sensitive region of the enzyme. In addition, k(cat) decreases for this mutant and the kinetic solvent isotope effect on k(cat), which was 2.0 in WT, is near unity in H48A. Despite being located 18 A from the enzyme active site, H48 is essential in coordinating the motions involved in the rate-limiting enzymatic step. These studies have identified, of approximately 160 potential exchangeable protons, a single site that is integral in the rate-limiting step in RNase A enzyme function.     

Structures of the N-terminal modules imply large domain motions during catalysis by methionine synthase

B(12)-dependent methionine synthase (MetH) is a large modular enzyme that utilizes the cobalamin cofactor as a methyl donor or acceptor in three separate reactions. Each methyl transfer occurs at a different substrate-binding domain and requires a different arrangement of modules. In the catalytic cycle, the cobalamin-binding domain carries methylcobalamin to the homocysteine (Hcy) domain to form methionine and returns cob(I)alamin to the folate (Fol) domain for remethylation by methyltetrahydrofolate (CH(3)-H(4)folate). Here, we describe crystal structures of a fragment of MetH from Thermotoga maritima comprising the domains that bind Hcy and CH(3)-H(4)folate. These substrate-binding domains are (beta alpha)(8) barrels packed tightly against one another with their barrel axes perpendicular. The properties of the domain interface suggest that the two barrels remain associated during catalysis. The Hcy and CH(3)-H(4)folate substrates are bound at the C termini of their respective barrels in orientations that position them for reaction with cobalamin, but the two active sites are separated by approximately 50 A. To complete the catalytic cycle, the cobalamin-binding domain must travel back and forth between these distant active sites.     

Cocatalytic zinc motifs in enzyme catalysis.

Cocatalytic zinc binding sites are characteristic of enzyme molecules which contain two or more zinc and/or other metal atoms. In each site an aspartate, glutamate, or histidine residue simultaneously binds to two zinc atoms or a zinc and a different metal atom. In the resultant amino acid bridge, two of the cocatalytic metal atoms bind to the same amino acid. Consequently the participating metal atoms are in close proximity and function as a catalytic unit, typical of this motif. In these functional units aspartate seems to be preferred over glutamate. Serine, threonine, tryptophan, and lysine residues are encountered as zinc ligands, although they have not so far been identified as ligands in monozinc enzymes or DNA-binding zinc proteins. The resultant coordination spheres and their mechanistic implications raise interesting questions for further study.     

The nature of enzyme catalysis in trypsin.

We present a combined quantum/molecular mechanical study of the trypsin-catalyzed hydrolysis of a specific tripeptide substrate, including the entire enzyme in the calculation, as well as 200 H2O molecules. The results illustrate how the enzyme and nearby H2O molecules stabilize the ionic intermediates in peptide hydrolysis, such that the reaction is calculated to have a barrier that is significantly smaller than the calculated and experimental base-catalyzed barrier of formamide hydrolysis in aqueous solution. This enables us to understand how serine proteases increase the rates for reactions that take place in their active sites, compared to the corresponding rates for analogous solution reactions.     

Large kinetic isotope effects in enzymatic proton transfer and the role of substrate oscillations

We propose an interpretation of the experimental findings of Klinman and coworkers [Cha, Y., Murray, C. J. & Klinman, J. P. (1989) Science 243, 1325–1330; Grant, K. L. & Klinman, J. P. (1989) Biochemistry 28, 6597–6605; and Bahnson, B. J. & Klinman, J. P. (1995) Methods Enzymol. 249, 373–397], who showed that proton transfer reactions that are catalyzed by bovine serum amine oxidase proceed through tunneling. We show that two different tunneling models are consistent with the experiments. In the first model, the proton tunnels from the ground state. The temperature dependence of the kinetic isotope effect is caused by a thermally excited substrate mode that modulates the barrier, as has been suggested by Borgis and Hynes [Borgis, D. & Hynes, J. T. (1991) J. Chem. Phys. 94, 3619–3628]. In the second model, there is both over-the-barrier transfer and tunneling from excited states. Finally, we propose two experiments that can distinguish between the possible mechanisms.     

Deuterium isotope effects in carcinogenesis by N-nitroso compounds

Summary A number of N-nitroso compounds and an azoxyalkane have been labeled with deuterium in various positions and have been administered to rats, hamsters, or mice in parallel with the unlabeled compounds. The treatments with the labeled and analogous unlabeled compounds were equimolar and for the same time. Mortality rates from tumors and tumor incidences were compared between deuteriumlabeled and the unlabeled analogs. In many cases more than one dose level was used for the comparisons. An increased rate of mortality from tumors or an increased incidence of induced tumors was considered an index of increased potency of one treatment compared with the other. Using these criteria deuterium in the alpha positions of nitrosodimethylamine, nitrosomorpholine, nitrosoheptamethyleneimine, and nitrosoazetidine reduced carcinogenic potency compared with the unlabeled compounds. This indicated that cleavage of a carbon-hydrogen bond in the alpha position was a rate-limiting step in carcinogenesis by these nitrosamines. In both nitrosomethylethylamine and nitroso-2,6-dimethylmorpholine, the presence of deuterium at different positions increased or decreased carcinogenic potency, suggesting that competition for oxidation between these sites might be the determining factor in activation of the molecule. This also applied to nitrosomethyl-n-butylamine and nitrosomethylphenylethylamine with deuterium at the methyl group or at the alpha carbon of the butyl or phenylethyl groups, and to azoxymethane with deuterium in the 1-methyl or 4-methyl group. In nitrosomethylcyclohexylamine, nitrosomethyl-n-dodecylamine, and dinitroso-2,6-dimethylpiperazine there was no detectable effect of deuterium on carcinogenic potency, suggesting that the conditions did not provide sufficient sensitivity for detection of an isotope effect, or that oxidation at the alpha carbon was not a rate-limiting step in carcinogenesis by these molecules.Research sponsored by the National Cancer Institute, DHHS, under contract No. N01-Co-23909 with Litton Bionetics, Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Helath and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government     

Platelet kinetic studies in patients with hyperlipoproteinemia: effects of clofibrate therapy.

Studies of platelet and fibrinogen kinetics in 27 patients with hyperlipoproteinemia and 28 control subjects demonstrated shortened platelet survival and increased platelet turnover in seven patients with type III and 10 patients with type IV-V hyperlipoproteinemia (p less than 0.01). There was no correlation between platelet survival time and specific lipid levels, vascular disease, sex or age. Platelet kinetics were not significantly altered from control values in eight patients with familial hypercholesterolemia. Platelet aggregation studies and fibrinogen kinetic measurements did not differ in any of the hyperlipoproteinemic groups of patients from those in control subjects. Despite significant changes in plasma lipids induced by clofibrate, platelet survival was significantly extended only in patients with type IV-V hyperlipoproteinemia (p less than 0.05). These results are consistent with the hypothesis that atherogenesis in patients with types III--V hyperlipoproteinemia may be associated with a process of endothelial desquamation, and type IIa hyperlipoproteinemia may involve nondesquamating endothelial injury.     

Nitrogen isotope effects induced by anammox bacteria

Nitrogen (N) isotope ratios (¹⁵N/¹⁴N) provide integrative constraints on the N inventory of the modern ocean. Anaerobic ammonium oxidation (anammox), which converts ammonium and nitrite to dinitrogen gas (N₂) and nitrate, is an important fixed N sink in marine ecosystems. We studied the so far unknown N isotope effects of anammox in batch culture experiments. Anammox preferentially removes ¹⁴N from the ammonium pool with an isotope effect of +23.5‰ to +29.1‰, depending on factors controlling reversibility. The N isotope effects during the conversion of nitrite to N₂ and nitrate are (i) inverse kinetic N isotope fractionation associated with the oxidation of nitrite to nitrate (−31.1 ± 3.9‰), (ii) normal kinetic N isotope fractionation during the reduction of nitrite to N₂ (+16.0 ± 4.5‰), and (iii) an equilibrium N isotope effect between nitrate and nitrite (−60.5 ± 1.0‰), induced when anammox is exposed to environmental stress, leading to the superposition of N isotope exchange effects upon kinetic N isotope fractionation. Our findings indicate that anammox may be responsible for the unresolved large N isotope offsets between nitrate and nitrite in oceanic oxygen minimum zones. Irrespective of the extent of N isotope exchange between nitrate and nitrite, N removed from the combined nitrite and nitrate (NO_x) pool is depleted in ¹⁵N relative to NO_x. This net N isotope effect by anammox is superimposed on the N isotope fractionation by the co-occurring reduction of nitrate to nitrite in suboxic waters, possibly enhancing the overall N isotope effect for N loss from oxygen minimum zones.The nitrogen (N) isotope effect associated with N loss pathways allows the isotopic tracing of N transformations in ocean waters and provides critical constraints on the global marine N budget (1–5). Culture studies have shown that heterotrophic denitrification exhibits a large N isotope effect (ε)^* of up to +30‰ (6–9). For decades, heterotrophic denitrification was the only known N loss pathway in the ocean (10). Consequently, strong enrichment of ¹⁵N in residual nitrite and nitrate (NO_x) from oxygen-deficient waters, as for example in the Eastern Tropical North Pacific and the Arabian Sea, was fully attributed to water column denitrification with an N isotope effect around +25‰ (2, 11). Recent studies have highlighted the significance of anaerobic ammonium oxidation (anammox) for regional N fluxes (12, 13), with possible ramifications regarding the global N balance (14, 15). However, N isotope effects associated with the anammox metabolism were unknown and therefore their potential impacts on the distribution of oceanic N isotopes could not be addressed. This lack of knowledge severely hampers the N-isotope–based assessment of the relative importance of water-column N loss compared with sedimentary N loss, the most poorly constrained flux in the marine combined nitrogen budget (16).     

High pressure studies of solvent effects on anthracene spectra

Measurements have been made of the effect of pressure on the peak location and peak shape for anthracene in a variety of liquid and plastic environments; both absorption and fluorescence studies were made. The results are discussed from two standpoints: in terms of the dielectric model and of a configuration coordinate model. For the latter model, the change of configuration coordinate (volume decrease of the system upon electronic excitation) is shown to correlate well with the product of the compressibility and polarizability of the solvent. For the dielectric model, it is found that the change of cavity volume with density is complex. However, the relative cavity volume obtained from emission measurements was consistently 10-15% smaller than that obtained from absorption. The cavity volume decreased with increasing polarizability of the solvent, and results obtained from absorption and emission were quite consistent in this regard.