Formation Of Covalent Intermediates Research Articles

Functional Analysis of DNMT3A DNA Methyltransferase Mutations Reported in Patients with Acute Myeloid Leukemia

In mammals, DNA methylation is necessary for the maintenance of genomic stability, gene expression regulation, and other processes. During malignant diseases progression, changes in both DNA methylation patterns and DNA methyltransferase (MTase) genes are observed. Human de novo MTase DNMT3A is most frequently mutated in acute myeloid leukemia (AML) with a striking prevalence of R882H mutation, which has been extensively studied. Here, we investigate the functional role of the missense mutations (S714C, R635W, R736H, R771L, P777R, and F752V) found in the catalytic domain of DNMT3A in AML patients. These were accordingly mutated in the murine Dnmt3a catalytic domain (S124C, R45W, R146H, R181L, P187R, and F162V) and in addition, one-site CpG-containing DNA substrates were used as a model system. The 3–15-fold decrease (S124C and P187R) or complete loss (F162V, R45W, and R146H) of Dnmt3a-CD methylation activity was observed. Remarkably, Pro 187 and Arg 146 are not located at or near the Dnmt3a functional motives. Regulatory protein Dnmt3L did not enhance the methylation activity of R45W, R146H, P187R, and F162V mutants. The key steps of the Dnmt3a-mediated methylation mechanism, including DNA binding and transient covalent intermediate formation, were examined. There was a complete loss of DNA-binding affinity for R45W located in the AdoMet binding region and for R146H. Dnmt3a mutants studied in vitro suggest functional impairment of DNMT3A during pathogenesis.

Quantum Chemical Study of Supercritical Carbon Dioxide Effects on Combustion Kinetics.

In oxy-fuel combustion, the pure oxygen (O2), diluted with CO2 is used as oxidant instead air. Hence, the combustion products (CO2 and H2O) are free from pollution by nitrogen oxides. Moreover, high pressures result in the near-liquid density of CO2 at supercritical state (sCO2). Unfortunately, the effects of sCO2 on the combustion kinetics are far from being understood. To assist in this understanding, in this work we are using quantum chemistry methods. Here we investigate potential energy surfaces of important combustion reactions in the presence of the carbon dioxide molecule. All transition states and reactant and product complexes are reported for three reactions: H2CO + HO2 → HCO + H2O2 (R1), 2HO2 → H2O2 + O2 (R2), and CO + OH → CO2 + H (R3). In reaction R3, covalent binding of CO2 to the OH radical and then the CO molecule opens a new pathway, including hydrogen transfer from oxygen to carbon atoms followed by CH bond dissociation. Compared to the bimolecular OH + CO mechanism, this pathway reduces the activation barrier by 5 kcal/mol and is expected to accelerate the reaction. In the case of hydroperoxyl self-reaction 2HO2 → H2O2 + O2 the intermediates, containing covalent bonds to CO2 are found not to be competitive. However, the spectator CO2 molecule can stabilize the cyclic transition state and lower the barrier by 3 kcal/mol. Formation of covalent intermediates is also discovered in the H2CO + HO2 → HCO + H2O2 reaction, but these species lead to substantially higher activation barriers, which makes them unlikely to play a role in hydrogen transfer kinetics. The van der Waals complexation with carbon dioxide also stabilizes the transition state and reduces the reaction barrier. These results indicate that the CO2 environment is likely to have a catalytic effect on combustion reactions, which needs to be included in kinetic combustion mechanisms in supercritical CO2.

Experimental Data in Support of a Direct Displacement Mechanism for Type I/II l-Asparaginases

Bacterial L-asparaginases play an important role in the treatment of certain types of blood cancers. We are exploring the guinea pig L-asparaginase (gpASNase1) as a potential replacement of the immunogenic bacterial enzymes. The exact mechanism used by L-asparaginases to catalyze the hydrolysis of asparagine into aspartic acid and ammonia has been recently put into question. Earlier experimental data suggested that the reaction proceeds via a covalent intermediate using a ping-pong mechanism, whereas recent computational work advocates the direct displacement of the amine by an activated water. To shed light on this controversy, we generated gpASNase1 mutants of conserved active site residues (T19A, T116A, T19A/T116A, K188M, and Y308F) suspected to play a role in hydrolysis. Using x-ray crystallography, we determined the crystal structures of the T19A, T116A, and K188M mutants soaked in asparagine. We also characterized their steady-state kinetic properties and analyzed the conversion of asparagine to aspartate using NMR. Our structures reveal bound asparagine in the active site that has unambiguously not formed a covalent intermediate. Kinetic and NMR assays detect significant residual activity for all of the mutants. Furthermore, no burst of ammonia production was observed that would indicate covalent intermediate formation and the presence of a ping-pong mechanism. Hence, despite using a variety of techniques, we were unable to obtain experimental evidence that would support the formation of a covalent intermediate. Consequently, our observations support a direct displacement rather than a ping-pong mechanism for l-asparaginases.

Acyltransferases in Bacteria

Long-chain-length hydrophobic acyl residues play a vital role in a multitude of essential biological structures and processes. They build the inner hydrophobic layers of biological membranes, are converted to intracellular storage compounds, and are used to modify protein properties or function as membrane anchors, to name only a few functions. Acyl thioesters are transferred by acyltransferases or transacylases to a variety of different substrates or are polymerized to lipophilic storage compounds. Lipases represent another important enzyme class dealing with fatty acyl chains; however, they cannot be regarded as acyltransferases in the strict sense. This review provides a detailed survey of the wide spectrum of bacterial acyltransferases and compares different enzyme families in regard to their catalytic mechanisms. On the basis of their studied or assumed mechanisms, most of the acyl-transferring enzymes can be divided into two groups. The majority of enzymes discussed in this review employ a conserved acyltransferase motif with an invariant histidine residue, followed by an acidic amino acid residue, and their catalytic mechanism is characterized by a noncovalent transition state. In contrast to that, lipases rely on completely different mechanism which employs a catalytic triad and functions via the formation of covalent intermediates. This is, for example, similar to the mechanism which has been suggested for polyester synthases. Consequently, although the presented enzyme types neither share homology nor have a common three-dimensional structure, and although they deal with greatly varying molecule structures, this variety is not reflected in their mechanisms, all of which rely on a catalytically active histidine residue.

Inhibition of murine DNA methyltransferase Dnmt3a by DNA duplexes containing pyrimidine-2(1H)-one

Here we studied the inhibition of the catalytic domain of Dnmt3a methyltransferase (Dnmt3a-CD) by DNA duplexes containing the mechanism-based inhibitor pyrimidine-2(1H)-one (P) instead of the target cytosine. It has been shown that conjugates of Dnmt3a-CD with P-DNA (DNA containing pyrimidine-2(1H)-one) are not stable to heating at 65°C in 0.1% SDS. The yield of covalent intermediate increases in the presence of the regulatory factor Dnmt3L. The importance of the DNA minor groove for covalent intermediate formation during the methylation reaction catalyzed by Dnmt3a-CD has been revealed. P-DNA was shown to inhibit Dnmt3a-CD; the IC(50) is 830nM. The competitive mechanism of inhibition of Dnmt3a-CD by P-DNA has been elucidated. It is suggested that therapeutic effect of zebularine could be achieved by inhibition of not only Dnmt1 but also Dnmt3a.

Stereospecific Proton Transfer by a Mobile Catalyst in Mammalian Fructose-1,6-bisphosphate Aldolase

Class I fructose-1,6-bisphosphate aldolases catalyze the interconversion between the enamine and iminium covalent enzymatic intermediates by stereospecific exchange of the pro(S) proton of the dihydroxyacetone-phosphate C3 carbon, an obligatory reaction step during substrate cleavage. To investigate the mechanism of stereospecific proton exchange, high resolution crystal structures of native and a mutant Lys(146) --> Met aldolase were solved in complex with dihydroxyacetone phosphate. The structural analysis revealed trapping of the enamine intermediate at Lys(229) in native aldolase. Mutation of conserved active site residue Lys(146) to Met drastically decreased activity and enabled trapping of the putative iminium intermediate in the crystal structure showing active site attachment by C-terminal residues 360-363. Attachment positions the conserved C-terminal Tyr(363) hydroxyl within 2.9A of the C3 carbon in the iminium in an orientation consistent with incipient re face proton transfer. We propose a catalytic mechanism by which the mobile C-terminal Tyr(363) is activated by the iminium phosphate via a structurally conserved water molecule to yield a transient phenate, whose developing negative charge is stabilized by a Lys(146) positive charge, and which abstracts the C3 pro(S) proton forming the enamine. An identical C-terminal binding mode observed in the presence of phosphate in the native structure corroborates Tyr(363) interaction with Lys(146) and is consistent with transient C terminus binding in the enamine. The absence of charge stabilization and of a mobile C-terminal catalyst explains the extraordinary stability of enamine intermediates in transaldolases.

Direct Detection and Kinetic Analysis of Covalent Intermediate Formation in the 4-Amino-4-deoxychorismate Synthase Catalyzed Reaction

Chorismate-utilizing enzymes catalyze diverse reactions, providing critical physiological functions unique to plants, bacteria, fungi, and some parasites. Their absence in animals makes them excellent targets for antimicrobials and herbicides. 4-Amino-4-deoxychorismate synthase (ADCS) catalyzes the first step in folate biosynthsis and shares a common core mechanism with isochorismate synthase (IS) and anthranilate synthase (AS), in which nucleophile addition at C2 initiates these reactions. Evidence was presented previously [He, Z., Stigers Lavoie, K. D., Bartlett, P. A., and Toney, M. D. (2004) J. Am. Chem. Soc. 126, 2378-2385] that K274 is the nucleophile in ADCS, implying formation of a covalent intermediate. Herein, we report the direct detection of this covalent intermediate formed in ADCS-catalyzed reactions by ESI-MS. Difference spectra show the covalent intermediate has an absorption maximum at 310 nm. This was used to study the pre-steady-state kinetics of covalent intermediate formation under various conditions. Additionally, E258 in ADCS was shown to be critical to formation of the covalent intermediate by acting as a general acid catalyst for loss of the C4 hydroxyl group. The E258A/D mutants both exhibit very low activity. Acetate is a poor chemical rescue agent for E258D but an excellent one for E258A, with a 20000-fold and 3000-fold rate increase for Gln-dependent and NH(4)(+)-dependent activities, respectively. Lastly, A213 in IS (structurally homologous to K274 in ADCS) was changed to lysine in an attempt to convert IS to an ADCS-like enzyme. HPLC studies support the formation of a covalent intermediate with this mutant.

Mechanism of Gly-Pro-pNA cleavage catalyzed by dipeptidyl peptidase-IV and its inhibition by saxagliptin (BMS-477118)

Dipeptidyl peptidase-IV (DPP-IV) is a serine protease with a signature Asp-His-Ser motif at the active site. Our pH data suggest that Gly-Pro-pNA cleavage catalyzed by DPP-IV is facilitated by an ionization of a residue with a p K of 7.2 ± 0.1. By analogy to other serine proteases this p K is suggestive of His-Asp assisted Ser addition to the P1 carbonyl carbon of the substrate to form a tetrahedral intermediate. Solvent kinetic isotope effect studies yielded a D 2 O k cat / K m = 2.9 ± 0.2 and a D 2 O k cat = 1.7 ± 0.2 suggesting that kinetically significant proton transfers contribute to rate limitation during acyl intermediate formation (leaving group release) and hydrolysis. A “burst” of product release during pre steady-state Gly-Pro- pNA cleavage indicated rate limitation in the deacylation half-reaction. Nevertheless, the amplitude of the burst exceeded the enzyme concentration significantly (∼15-fold), which is consistent with a branching deacylation step. All of these data allowed us to better understand DPP-IV inhibition by saxagliptin (BMS-477118). We propose a two-step inhibition mechanism wherein an initial encounter complex is followed by covalent intermediate formation. Final inhibitory complex assembly ( k on) depends upon the ionization of an enzyme residue with a p K of 6.2 ± 0.1, and we assigned it to the catalytic His-Asp pair which enhances Ser nucleophilicity for covalent addition. An ionization with a p K of 7.9 ± 0.2 likely reflects the P2 terminal amine of the inhibitor hydrogen bonding to Glu205/Glu206 in the enzyme active site. The formation of the covalent enzyme–inhibitor complex was reversible and dissociated with a k off of (5.5 ± 0.4) × 10 −5 s −1, thus yielding a K i ∗ (as k off/ k on) of 0.35 nM, which is in good agreement with the value of 0.6 nM obtained from steady-state inhibition studies. Proton NMR spectra of DPP-IV showed a downfield resonance at 16.1 ppm. Two additional peaks in the 1H NMR spectra at 17.4 and 14.1 ppm were observed upon mixing the enzyme with saxagliptin. Fractionation factors ( ϕ) of 0.6 and 0.5 for the 17.4 and 14.1 ppm peaks, respectively, are suggestive of short strong hydrogen bonds in the enzyme–inhibitor complex.

Solution Structures and Backbone Dynamics of Arsenate Reductase from Bacillus subtilis: REVERSIBLE CONFORMATIONAL SWITCH ASSOCIATED WITH ARSENATE REDUCTION

Arsenate reductase encoded by the chromosomal arsC gene in Bacillus subtilis catalyzes the intracellular reduction of arsenate to arsenite, which is then extruded from cells through an efficient and specific transport system. Herein, we present the solution structures and backbone dynamics of both the reduced and oxidized forms of arsenate reductase from B. subtilis. The overall structures of both forms are similar to those of bovine low molecular weight protein-tyrosine phosphatase and arsenate reductase from Staphylococcus aureus. However, several features of the tertiary structure and mobility are notably different between the reduced and oxidized forms of B. subtilis arsenate reductase, particularly in the P-loop region and the segment Cys(82)-Cys(89). The backbone dynamics results demonstrated that the reduced form of arsenate reductase undergoes millisecond conformational changes in the functional P-loop and Cys(82)-Cys(89), which may facilitate the formation of covalent intermediates and subsequent reduction of arsenate. In the oxidized form, Cys(82)-Cys(89) shows motional flexibility on both picosecond-to-nanosecond and possibly millisecond time scales, which may facilitate the reduction of the oxidized enzyme by thioredoxin to regenerate the active enzyme. Overall, the internal dynamics and static structures of the enzyme provide insights into the molecular mechanism of arsenate reduction, especially the reversible conformational switch and changes in internal motions associated with the catalytic reaction.

Covalent Intermediates and Enzyme Proficiency

Large enzyme efficiencies are due to small rate constants for the reaction in water, not to the formation of covalent intermediates.

Catalytic Mechanism of DNA-(cytosine-C5)-methyltransferases Revisited: Covalent Intermediate Formation is not Essential for Methyl Group Transfer by the Murine Dnmt3a Enzyme

Co-transfections of reporter plasmids and plasmids encoding the catalytic domain of the murine Dnmt3a DNA methyltransferase lead to inhibition of reporter gene expression. As Dnmt3a mutants with C→A and E→A exchanges in the conserved PCQ and ENV motifs in the catalytic center of the enzyme also cause repression, we checked for their catalytic activity in vitro. Surprisingly, the activity of the cysteine variant and of the corresponding full-length Dnmt3a variant is only two to sixfold reduced with respect to wild-type Dnmt3a. In contrast, enzyme variants carrying E→A, E→D or E→Q exchanges of the ENV glutamate are catalytically almost inactive, demonstrating that this residue has a central function in catalysis. Since the glutamic acid residue contacts the flipped base, its main function could be to hold the target base at a position that supports methyl group transfer. Whereas wild-type Dnmt3a and the ENV variants form covalent complexes with 5-fluorocytidine modified DNA, the PCN variant does not. Therefore, covalent complex formation is not essential in the reaction mechanism of Dnmt3a. We propose that correct positioning of the flipped base and the cofactor and binding to the transition state of methyl group transfer are the most important roles of the Dnmt3a enzyme in the catalytic cycle of methyl group transfer.

Mechanism-based inactivation of γ-aminobutyric acid aminotransferase by 3-amino-4-fluorobutanoic acid

The mechanism of inactivation of the pyridoxal 5′-phosphate (PLP)-dependent enzyme γ-aminobutyric acid (GABA) aminotransferase by 3-amino-4-fluorobutanoic acid ( 2) has been investigated. As in the case of the homologue, 4-amino-5-fluoropentanoic acid ( 1), 2 equiv of radiolabeled inactivator become covalently attached to the enzyme, and no transamination, as determined by the lack of conversion of [1- 14C] α-ketoglutarate into [1- 13C] glutamate during inactivation, was observed. In the case of 1, the conclusion was that inactivation was completely the result of modification of the coenzyme and that there was no metabolic turnover; every enzyme molecule catalysed the conversion of one molecule of inactivator to the activated species, which inactivated the enzyme by an enamine mechanism. With 2, however, 6.7±0.7 equiv of fluoride ions were released during inactivation, and it took 7.6±0.7 inactivator molecules to inactivate each enzyme dimer. Since no transamination was occurring, another metabolic event besides inactivation must result from the PLP form of the enzyme. Inactivation of GABA amino-transferase with [1,2- 14C]- 2 produced [ 14C] acetoacetic acid (about 5.5 equiv) as the metabolite. The 1.93±0.25 equiv of radioactivity covalently bound to the enzyme after inactivation with [1,2- 14C]- 2 and gel filtration were completely released by base treatment. HPLC analysis showed that three radioactive compounds, identified as 2, the product of reaction of PLP with acetone ( 3), and the product of reaction of PLP with acetoacetate ( 4), were detected. The release of 3 and 4 and the prevention of release of radioactivity by treatment with sodium borohydride are consistent with the formation of covalent intermediates that have β-carbonyl-like character, such as 6 and/or 7 (Scheme 2). Inactivation of [ 3H] PLP-reconstituted GABA aminotransferase with 2 followed by gel filtration then base denaturation released all of the radioactivity as a mixture of PLP, 3, and 4. Inactivation with [1,2- 14C]- 2 resulted in the release of 1.37 equiv of 14CO 2, which was shown to be the result of decarboxylation of the acetoacetate/ 4 after release from the enzyme. These results are not consistent with a Michael addition mechanism (Scheme 3), but are consistent with inactivation by an enamine mechanism; release of the enamine five out of seven turnovers accounts for the formation of acetoacetate as the metabolite. To account for the detection of PLP and 2 after denaturation, it is suggested that a nonproductive formation of the Schiff base of PLP with 2 occurs in the second subunit of the enzyme; this complex is released and hydrolysed to PLP and 2 upon base denaturation.

Human cathepsin O. Molecular cloning from a breast carcinoma, production of the active enzyme in Escherichia coli, and expression analysis in human tissues.

A cDNA encoding a novel member of the cysteine proteinase family of proteins has been cloned from a human breast carcinoma cDNA library, by using a polymerase chain reaction-based cloning strategy. The isolated cDNA contains an open reading frame coding for a polypeptide of 321 amino acids that has been tentatively called cathepsin O. This protein presents all the structural features characteristic of the different cysteine proteinases identified to date, including the active site cysteine residue that is involved in covalent intermediate formation during peptide hydrolysis. The cathepsin O cDNA was expressed in Escherichia coli, and after purification and refolding, the recombinant protein was able to degrade the synthetic peptides benzyloxycarbonyl-Phe-Arg-7-amido-4- methylcoumarin and benzyloxycarbonyl-Arg-Arg-7-amido-4-methylcoumarin widely used as substrates for cysteine proteinases. Cathepsin O proteolytic activity was abolished by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), an inhibitor of this subclass of proteolytic enzymes, thus providing additional evidence that the isolated cDNA codes for an authentic cysteine proteinase. Northern blot analysis of poly(A)+ RNAs isolated from a variety of human tissues demonstrated that cathepsin O is expressed in all examined tissues, which is consistent with a putative role of this protein as a proteolytic enzyme involved in normal cellular protein degradation and turnover.

Probing conformational states of spin‐labeled aspartate aminotransferase by ESR

Mitochondrial aspartate aminotransferase was selectively labeled with various maleimide-linked nitroxide spin labels at the conformationally sensitive Cys166. The mobility of the spin group was found to increase with increasing length of the spacer between the nitroxide and maleimide moiety. The label with the ethylcarbamoyl group, a spacer of intermediate length, responded sensitively to conformational changes of aspartate aminotransferase. The modification with this label decreased the enzymic activity to 30% of its initial value and increased the affinity for various substrates and inhibitors 5-10-fold. Identical ESR spectra were obtained for the pyridoxal and pyridoxamine form of the enzyme. These spectra are complex, consisting of an isotropic and at least two anisotropic components. The spectral complexity is attributed to different modes of interaction of the spin label with its local protein environment giving rise to different motional states. The same changes in the ESR spectra have been observed upon formation of the adsorption complex of the pyridoxal form with a competitive inhibitor and on formation of covalent intermediates of the transamination reaction. Essentially, the isotropic component is converted to a new anisotropic one as the local environment changes due to a conformational adaptation of aspartate aminotransferase. The ESR data are consistent with an equilibrium between two conformational states of the enzyme but inconsistent with individual protein conformations of the various intermediates of the transamination reaction. The two conformational states may be assigned to the open and closed conformations as defined by X-ray crystallography. In the adsorption complex of the pyridoxal enzyme, and in the covalent intermediates, the two-state equilibrium appears to be shifted towards the closed conformation in which the spin label is more rigidly bound, as also suggested by molecular dynamic simulations of the label modelled into aspartate aminotransferase. In contrast the formation of adsorption complexes between the pyridoxamine form and aspartate or maleate was not accompanied by the same shift of the conformational equilibrium.

Mechanism of Enzymatic and Nonenzymatic Prolyl Cis-Trans Isomerization

This chapter describes the mechanism of both enzymatic and non-enzymatic prolyl isomerization. Non-enzymatic prolyl isomerization and rotation about amide bonds are reviewed. Amide rotation is mechanistically related to prolyl isomerization and serves as a useful model for prolyl isomerization. A common mechanism for these reactions and a structure for the rate-limiting transition state are reviewed. The chapter also discusses on what is known of enzyme-catalyzed prolyl isomerization. The chemistry of non-enzymatic prolyl isomerizations and the mechanisms of the non-enzymatic catalysis of this reaction dictate the catalytic strategy that is used by prolyl isomerases. Finally, it leads to a mechanistic proposal for enzymatic prolyl isomerization. The catalytic strategy that an enzyme develops over the evolutionary time is dictated by the chemistry of the reaction being catalyzed. The prolyl isomerases are able to stabilize the non-enzymatic transition state without formation of covalent intermediates. Based on a k cat value of 10 4 sec -1 for CyP and a k uncut of 10 -2 sec -1 for the cis -to-trans isomerization of Suc-Ala-Ala- cis- Pro-Phe-pNA, an acceleration factor, k cat /k uncut of 10 6 , is calculated that corresponds to a transition state stabilization free energy of over 8 kcal/mol 3 . The energetic price to distort the Xaa-Pro bond out of planarity must ultimately be paid by favorable transition state interactions.

Cryoenzymology of porcine pepsin.

Physical and kinetic properties of porcine pepsin have been examined in aqueous methanol solvents at temperatures below ambient to seek evidence for covalent intermediates in the catalyzed hydrolysis of good substrates. It was first demonstrated that aqueous methanol cryosolvents have no significant deleterious effects upon this protein. The addition of methanol does lead to a drastic reduction in the midpoint of the thermal melting curve of pepsin. This could account for rate reductions previously observed in catalysis by this enzyme. This effect is lessened by the addition of active-site ligands including substrates and is fully reversible upon dilution into aqueous solution. Two substrates were chosen which have chromophoric groups on opposite sides of the scissile peptide bond. The UV spectral changes from hydrolysis of Pro-Thr-Glu-Phe-(NO2)Phe-Arg-Leu and the fluorescence spectral changes from hydrolysis of DNS-Ala-Ala-Phe-Phe-OP4P+-CH3 were studied at temperatures down to -60 degrees C. The resulting Arrhenius plots were linear in the region where pepsin exists in the native state with downward curvature exhibited at higher temperatures where the reversible denaturation occurs. No "burst" reactions were observed with either substrate. In addition, efforts at trapping intermediates by low-temperature denaturation and precipitation have provided no evidence for covalent intermediates on the reaction pathway. Although this evidence is negative, we cannot rule out the possibility of the formation of covalent intermediates following an initial rate-limiting step.

Question of covalent intermediate formation in the flavine-catalyzed carbonyl to carbinol oxidation-reduction reaction

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTQuestion of covalent intermediate formation in the flavine-catalyzed carbonyl to carbinol oxidation-reduction reactionSeiji. Shinkai and Thomas C. BruiceCite this: J. Am. Chem. Soc. 1973, 95, 22, 7526–7528Publication Date (Print):October 1, 1973Publication History Published online1 May 2002Published inissue 1 October 1973https://doi.org/10.1021/ja00803a064RIGHTS & PERMISSIONSArticle Views69Altmetric-Citations23LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (351 KB) Get e-Alerts Get e-Alerts