The first example of molecularly imprinted nanogels with aldolase type I activity

The molecular-imprinting approach was used to obtain a nanogel preparation capable of catalysing the cross-aldol reaction between 4-nitrobenzaldehyde and acetone. A polymerisable proline derivative was used as the functional monomer to mimic the enamine-based mechanism of aldolase type I enzymes. The diketone template used to create the cavity was designed to imitate the intermediate of the aldol reaction and was bound to the functional monomer using a reversible covalent interaction prior to polymerisation. By using a high-dilution polymerisation method, soluble imprinted nanogels were prepared with dimensions similar to those of an enzyme and with the advantage of solubility and flexibility previously unattainable with monolithic polymers. Following template removal and estimation of active-site concentrations, the kinetic characterisation of both imprinted and non-imprinted nanogels was carried out with catalyst concentrations between 0.7 and 3.5 mol %. Imprinted nanogel AS147 was found to have a k(cat) value of 0.25 x 10(-2) min(-1), the highest value ever achieved with imprinted polymers catalysing C--C bond formation. Comparison of the catalytic constants for both imprinted nanogel AS147 and non-imprinted nanogel AS133 gave a ratio of k(cat 147)/k(cat 133)=18.8, which is indicative of good imprinting efficiency and highlights the significance of the template during the imprinting process. This work represents a significant demonstration of the superiority of nanogels, when the molecular-imprinting approach is used, over "bulk" polymers for the generation of catalysts.     

Functional mimicry of carboxypeptidase A by a combination of transition state stabilization and a defined orientation of catalytic moieties in molecularly imprinted polymers

An artificial model for the natural enzyme carboxypeptidase A has been constructed by molecular imprinting in synthetic polymers. The tetrahedral transition state analogues (TSAs 4 and 5) for the carbonate hydrolysis have been designed as templates to allow incorporation of the main catalytic elements, an amidinium group and a Zn(2+) or Cu(2+) center, in a defined orientation in the transition state imprinted active site. The complexation of the functional monomer and the template in presence of Cu(2+) through stoichiometric noncovalent interaction was established on the basis of (1)H NMR studies and potentiometric titration. The Cu(2+) center was introduced into the imprinted cavity during polymerization or by substitution of Zn(2+) in Zn(2+) imprinted polymers. The direct introduction displayed obvious advantages in promoting catalytic efficiency. With substrates exhibiting a very similar structure to the template, an extraordinarily high enhancement of the rate of catalyzed to uncatalyzed reaction (k(cat)/k(uncat)) of 10(5)-fold was observed. If two amidinium moieties are introduced in proximity to one Cu(2+) center in the imprinted cavity by complexation of the functional monomer 3 with the template 5, the imprinted catalysts exhibited even higher activities and efficiencies for the carbonate hydrolysis with k(cat)/k(uncat) as high as 410,000. These are by far the highest values obtained for molecularly imprinted catalysts, and they are also considerably higher compared to catalytic antibodies. Our kinetic studies and competitive inhibition experiments with the TSA template showed a clear indication of a very efficient imprinting procedure. In addition, this demonstrates the important role of the transition state stabilization during the catalysis of this reaction.     

RNA-cleaving DNA enzymes with altered regio- or enantioselectivity

In vitro evolution methods were used to obtain DNA enzymes that cleave either a 2',5'-phosphodiester following a D-ribonucleotide or a 3',5'-phosphodiester following an L-ribonucleotide. Both enzymes can operate in an intermolecular reaction format with multiple turnover. The DNA enzyme that cleaves a 2',5'-phosphodiester exhibits a k(cat) of approximately 0.01 min(-1) and catalytic efficiency, k(cat)/K(m), of approximately 10(8) M(-1) min(-1). The enzyme that cleaves an L-ribonucleotide is about 10-fold slower and has a catalytic efficiency of approximately 4 x 10(5) M(-1) min(-1). Both enzymes require a divalent metal cation for their activity and have optimal catalytic rate at pH 7-8 and 35-50 degrees C. In a comparison of each enzyme's activity with either its corresponding substrate that contains an unnatural ribonucleotide or a substrate that instead contains a standard ribonucleotide, the 2',5'-phosphodiester-cleaving DNA enzyme exhibited a regioselectivity of 6000-fold, while the L-ribonucleotide-cleaving DNA enzyme exhibited an enantioselectivity of 40-fold. These molecules demonstrate how in vitro evolution can be used to obtain regio- and enantioselective catalysts that exhibit specificities for nonnatural analogues of biological compounds.     

Remarkable remote chiral recognition in a reaction mediated by a catalytic antibody

The crystal structures of catalytic antibody D2.3 Fab with the two enantiomers, 7D and 7L, which represent transition state analogues for the hydrolysis of the corresponding esters, 6D and 6L, were determined to better understand remarkable reactivity differences: the L-ester displayed significantly tighter binding (K(M)) and increased catalytic activity (k(cat)) with D2.3, even though the chiral center is 7 bonds distant from the reaction center. Surprisingly, the electron densities of the liganded phosphonates, 7D and 7L, within the D2.3 binding/reaction site were essentially identical, highlighting the subtle influences of protein interactions on chemical behavior.     

Accumulation of tetrahedral intermediates in cholinesterase catalysis: a secondary isotope effect study

In a previous communication, kinetic β-deuterium secondary isotope effects were reported that support a mechanism for substrate-activated turnover of acetylthiocholine by human butyrylcholinesterase (BuChE) wherein the accumulating reactant state is a tetrahedral intermediate ( Tormos , J. R. ; et al. J. Am. Chem. Soc. 2005 , 127 , 14538 - 14539 ). In this contribution additional isotope effect experiments are described with acetyl-labeled acetylthiocholines (CL(3)COSCH(2)CH(2)N(+)Me(3); L = H or D) that also support accumulation of the tetrahedral intermediate in Drosophila melanogaster acetylcholinesterase (DmAChE) catalysis. In contrast to the aforementioned BuChE-catalyzed reaction, for this reaction the dependence of initial rates on substrate concentration is marked by pronounced substrate inhibition at high substrate concentrations. Moreover, kinetic β-deuterium secondary isotope effects for turnover of acetylthiocholine depended on substrate concentration, and gave the following: (D3)k(cat)/K(m) = 0.95 ± 0.03, (D3)k(cat) = 1.12 ± 0.02 and (D3)βk(cat) = 0.97 ± 0.04. The inverse isotope effect on k(cat)/K(m) is consistent with conversion of the sp(2)-hybridized substrate carbonyl in the E + A reactant state into a quasi-tetrahedral transition state in the acylation stage of catalysis, whereas the markedly normal isotope effect on k(cat) is consistent with hybridization change from sp(3) toward sp(2) as the reactant state for deacylation is converted into the subsequent transition state. Transition states for Drosophila melanogaster AChE-catalyzed hydrolysis of acetylthiocholine were further characterized by measuring solvent isotope effects and determining proton inventories. These experiments indicated that the transition state for rate-determining decomposition of the tetrahedral intermediate is stabilized by multiple protonic interactions. Finally, a simple model is proposed for the contribution that tetrahedral intermediate stabilization provides to the catalytic power of acetylcholinesterase.     

Modulation of the catalytic behavior of alpha-chymotrypsin at monolayer-protected nanoparticle surfaces

Amino-acid-functionalized gold clusters modulate the catalytic behavior of alpha-chymotrypsin (ChT) toward cationic, neutral, and anionic substrates. Kinetic studies reveal that the substrate specificity (k(cat)/K(M)) of ChT-nanoparticle complexes increases by approximately 3-fold for the cationic substrate but decreases by 95% for the anionic substrate as compared with that of free ChT, providing enhanced substrate selectivity. Concurrently, the catalytic constants (k(cat)) of ChT show slight augmentation for the cationic substrate and significant attenuation for the anionic substrate in the presence of amino-acid-functionalized nanoparticles. The amino acid monolayer on the nanoparticle is proposed to control both the capture of substrate by the active site and release of product through electrostatic interactions, leading to the observed substrate specificities and catalytic constants.     

Beta-D-xylosidase from Selenomonas ruminantium: thermodynamics of enzyme-catalyzed and noncatalyzed reactions

Beta-D-Xylosidase/alpha-L-arabinofuranosidase from Selenomonas ruminantium is the most active enzyme known for catalyzing hydrolysis of 1,4-beta-D: -xylooligosaccharides to D-xylose. Temperature dependence for hydrolysis of 4-nitrophenyl-beta-D-xylopyranoside (4NPX), 4-nitrophenyl-alpha-L-arabinofuranoside (4NPA), and 1,4-beta-D-xylobiose (X2) was determined on and off (k (non)) the enzyme at pH 5.3, which lies in the pH-independent region for k (cat) and k (non). Rate enhancements (k (cat)/k (non)) for 4NPX, 4NPA, and X2 are 4.3 x 10(11), 2.4 x 10(9), and 3.7 x 10(12), respectively, at 25 degrees C and increase with decreasing temperature. Relative parameters k (cat) (4NPX)/k (cat) (4NPA), k (cat) (4NPX)/k (cat) (X2), and (k (cat)/K (m))(4NPX)/(k (cat)/K (m))(X2) increase and (k (cat)/K (m))(4NPX)/(k (cat)/K (m))(4NPA), (1/K (m))(4NPX)/(1/K (m))(4NPA), and (1/K (m))(4NPX)/(1/K (m))(X2) decrease with increasing temperature.     

Proton inventory studies of alpha-thrombin-catalyzed reactions of substrates with selected P and P' sites

Deuterium kinetic solvent isotope effects for the human alpha-thrombin-catalyzed hydrolysis of (1) substrates with selected P(1)-P(3) sites, Z-Pro-Arg-7-amido-4-methylcoumarin (7-AMC), N-t-Boc-Val-Pro-Arg-7-AMC, Bz-Phe-Val-Arg-4-nitroanilide (pNA), and H-D-Phe-L-Pip-Arg-pNA, are (DOD)k(cat) = (2.8-3.3) +/- 0.1 and (DOD)(k(cat)/K(m)) = (0.8-2.1) +/- 0.1 and (2) internally fluorescence-quenched substrates (a) (AB)Val-Phe-Pro-Arg-Ser-Phe-Arg-Leu-Lys(DNP)-Asp-OH, an optimal sequence, and (b) (AB)Val-Ser-Pro-Arg-Ser-Phe-Gln-Lys(DNP)-Asp-OH, recognition sequence for factor VIII, are (DOD)k(cat) = 2.2 +/- 0.2 and (DOD)(k(cat)/K(m)) = (0.8-0.9) +/- 0.1, at the pL (L = H, D) maximum, 8.4-9.0, and (25.0-26.0) +/- 0.1 degrees C. The most plausible models fitting the partial isotope effect (proton inventory) data have been selected on the basis of lowest values of the reduced chi squared and consistency of fractionation factors at all substrate concentrations, assuming rate-determining acylation. The data for Z-Pro-Arg-7-AMC are consistent with a single-proton bridge at the transition state phi(TS) = 0.39 +/- 0.05 and components for solvent reorganization phi(S) = 0.8 +/- 0.1 and phi(S) = 1.22 for k(cat) and k(cat)/K(m), respectively. The data for tripeptide amides fit bowl-shaped curves; an example is N-t-Boc-Val-Pro-Arg-7-AMC: phi(TS)(1) = phi(TS)(2) = 0.57 +/- 0.01 and phi(S) = 1 for k(cat) and 1.6 +/- 0.1 for k(cat)/K(m). Proton inventories for the nonapeptide (2b) are linear. The data for k(cat) for H-D-Phe-L-Pip-Arg-pNA and the decapeptide (2a) are most consistent with two identical fractionation factors for catalytic proton bridging, phi(TS)(1) = phi(TS)(2) = 0.68 +/- 0.02 and a large inverse component (phi(S) = 3.1 +/- 0.5) for the latter, indicative of substantial solvent reorganization upon leaving group departure. Proton inventory curves for k(cat)/K(m) for nearly all substrates are dome-shaped with an inverse isotope effect component (phi(S) = 1.2-2.4) originating from solvent reorganization during association of thrombin with substrate. These large contributions from medium effects are in full accord with the conformational adjustments required for the fulfillment of the dual, hemostatic and thrombolytic, functions of thrombin.     

Quantitative analysis of the effect of salt concentration on enzymatic catalysis.

Like pH, salt concentration can have a dramatic effect on enzymatic catalysis. Here, a general equation is derived for the quantitative analysis of salt-rate profiles: k(cat)/K(M) = (k(cat)/K(M))(MAX)/[1+([Na+]/K[Na+])(n')], where (k(cat)/K(M))(MAX) is the physical limit of k(cat)/K(M), K(Na+) is the salt concentration at which k(cat)/K(M) = (k(cat)/K(M))(MAX)/2, and -n' is the slope of the linear region in a plot of log(k(cat)/K(M)) versus log [Na+]. The value of n' is of special utility, as it reflects the contribution of Coulombic interactions to the uniform binding of the bound states. This equation was used to analyze salt effects on catalysis by ribonuclease A (RNase A), which is a cationic enzyme that catalyzes the cleavage of an anionic substrate, RNA, with k(cat)/K(M) values that can exceed 10(9) M(-1) s(-1). Lys7, Arg10, and Lys66 comprise enzymic subsites that are remote from the active site. Replacing Lys7, Arg10, and Lys66 with alanine decreases the charge on the enzyme as well as the value of n'. Likewise, decreasing the number of phosphoryl groups in the substrate decreases the value of n'. Replacing Lys41, a key active-site residue, with arginine creates a catalyst that is limited by the chemical conversion of substrate to product. This change increases the value of n', as expected for a catalyst that is more sensitive to changes in the binding of the chemical transition state. Hence, the quantitative analysis of salt-rate profiles can provide valuable insight into the role of Coulombic interactions in enzymatic catalysis.     

Preparation of surface molecularly imprinted Ru-complex catalysts for asymmetric transfer hydrogenation in water media

Molecularly imprinted Ru-complex catalysts acting in water were prepared on a SiO(2) surface by molecular imprinting of a SiO(2)-supported Ru-complex using organic polymers as surface matrix overlayers. (R)-1-(o-fluorophenyl)ethanol, which is one of the hydrogenated products of o-fluoroacetophenone, was imprinted on the supported Ru-complex as a template, and an active Ru-complex with a shape-selective reaction space (molecularly imprinted cavity) was prepared inside the wall of the hydrophobic organic polymer matrix overlayers. Structures of the SiO(2)-supported and molecularly imprinted Ru catalysts were characterized by means of solid-state NMR, XPS, XRF, ICP, UV/vis, XAFS, TGA, and SEM. The molecularly imprinted Ru catalysts exhibited fine shape selectivity and enantioselectivity for the asymmetric transfer hydrogenation of o-fluoroacetophenone and its derivatives.     

Purification and characterization of a glycoside hydrolase family 43 beta-xylosidase from Geobacillus thermoleovorans IT-08

The gene encoding a glycoside hydrolase family 43 beta-xylosidase (GbtXyl43A) from the thermophilic bacterium Geobacillus thermoleovorans strain IT-08 was synthesized and cloned with a C-terminal His-tag into a pET29b expression vector. The recombinant gene product termed GbtXyl43A was expressed in Escherichia coli and purified to apparent homogeneity. Michaelis-Menten kinetic parameters were obtained for the artificial substrates p-nitrophenyl-beta-D: -xylopyranose (4NPX) and p-nitrophenyl-alpha-L: -arabinofuranose (4NPA), and it was found that the ratio k (cat)/K (m) 4NPA/k (cat)/K (m) 4NPX was approximately 7, indicting greater catalytic efficiency for 4NP hydrolysis from the arabinofuranose aglycon moiety. Substrate inhibition was observed for the substrates 4-methylumbelliferyl xylopyranoside (muX) and the arabinofuranoside cogener (muA), and the ratio k (cat)/K (m) muA/k (cat)/K (m) muX was approximately 5. The enzyme was competitively inhibited by monosaccharides, with an arabinose K (i) of 6.8 +/- 0.62 mM and xylose K (i) of 76 +/- 8.5 mM. The pH maxima was 5.0, and the enzyme was not thermally stable above 54 degrees C, with a t (1/2) of 35 min at 57.5 degrees C. GbtXyl43A showed a broad substrate specificity for hydrolysis of xylooligosaccharides up to the highest degree of polymerization tested (xylopentaose), and also released xylose from birch and beechwood arabinoxylan.     

A promiscuous glutathione transferase transformed into a selective thiolester hydrolase

Human glutathione transferase A1-1 (hGST A1-1) can be reengineered by rational design into a catalyst for thiolester hydrolysis with a catalytic proficiency of 1.4 x 10(7) M(-1). The thiolester hydrolase, A216H that was obtained by the introduction of a single histidine residue at position 216 catalyzed the hydrolysis of a substrate termed GSB, a thiolester of glutathione and benzoic acid. Here we investigate the substrate requirements of this designed enzyme by screening a thiolester library. We found that only two thiolesters out of 18 were substrates for A216H. The A216H-catalyzed hydrolysis of GS-2 (thiolester of glutathione and naphthalenecarboxylic acid) exhibits a k(cat) of 0.0032 min(-1) and a KM of 41 microM. The previously reported catalysis of GSB has a k(cat) of 0.00078 min(-1) and KM of 5 microM. The k(cat) for A216H-catalyzed hydrolysis of GS-2 is thus 4.1 times higher than for GSB. The catalytic proficiency (k(cat)/KM)/k(uncat) for GS-2 is 3 x 10(6) M(-1). The promiscuous feature of the wt protein towards a range of different substrates has not been conserved in A216H but we have obtained a selective enzyme with high demands on the substrate.     

Polyethylene imine derivatives ('synzymes') accelerate phosphate transfer in the absence of metal

The efficient integration of binding, catalysis, and multiple turnovers remains a challenge in building enzyme models. We report that systematic derivatization of polyethylene imine (PEI) with alkyl (C(2)-C(12)), benzyl, and guanidinium groups gives rise to catalysts ('synzymes') with rate accelerations (k(cat)/k(uncat)) of up to 10(4) for the intramolecular transesterification of 2-hydroxypropyl-p-nitrophenyl phosphate, HPNP, in the absence of metal. The synzymes exhibit saturation kinetics (K(M) approximately 250 microM, k(cat) approximately 0.5 min(-1)) and up to 2340 turnovers per polymer molecule. Catalysis can be specifically and competitively inhibited by anionic and hydrophobic small molecules. The efficacy of catalysis is determined by the PEI derivatization pattern. The derivatization reagents exert a synergistic effect, i.e., their combinations increase catalysis by more than the sum of each single modification. The pH-rate profile for k(cat)/K(M) is bell shaped with a maximum at pH 7.85 and can be explained as a combination of two effects that both have to be operative for optimal activity: K(M) increases at high pH due to deprotonation of PEI amines that bind the anionic substrate and kcat decreases as the availability of hydroxide decreases at low pH. Thus, catalysis is based on substrate binding by positively charged amine groups and the presence of hydroxide ion in active sites in an environment that is tuned for efficient catalysis. Inhibition studies suggest that the basis of catalysis and multiple turnovers is differential molecular recognition of the doubly negatively charged transition state (over singly charged ground state and product): this contributes a factor of at least 5-10-fold to catalysis and product release.     

Design of a dinuclear nickel(II) bioinspired hydrolase to bind covalently to silica surfaces: synthesis, magnetism, and reactivity studies

Presented herein is the design of a dinuclear Ni(II) synthetic hydrolase [Ni(2)(HBPPAMFF)(μ-OAc)(2)(H(2)O)]BPh(4) (1) (H(2)BPPAMFF = 2-[(N-benzyl-N-2-pyridylmethylamine)]-4-methyl-6-[N-(2-pyridylmethyl)aminomethyl)])-4-methyl-6-formylphenol) to be covalently attached to silica surfaces, while maintaining its catalytic activity. An aldehyde-containing ligand (H(2)BPPAMFF) provides a reactive functional group that can serve as a cross-linking group to bind the complex to an organoalkoxysilane and later to the silica surfaces or directly to amino-modified surfaces. The dinuclear Ni(II) complex covalently attached to the silica surfaces was fully characterized by different techniques. The catalytic turnover number (k(cat)) of the immobilized Ni(II)Ni(II) catalyst in the hydrolysis of 2,4-bis(dinitrophenyl)phosphate is comparable to the homogeneous reaction; however, the catalyst interaction with the support enhanced the substrate to complex association constant, and consequently, the catalytic efficiency (E = k(cat)/K(M)) and the supported catalyst can be reused for subsequent diester hydrolysis reactions.     

Inhibitor scaffolds as new allele specific kinase substrates

The elucidation of protein kinase signaling networks is challenging due to the large size of the protein kinase superfamily (>500 human kinases). Here we describe a new class of orthogonal triphosphate substrate analogues for the direct labeling of analogue-specific kinase protein targets. These analogues were constructed as derivatives of the Src family kinase inhibitor PP1 and were designed based on the crystal structures of PP1 bound to HCK and N(6)-(benzyl)-ADP bound to c-Src (T338G). 3-Benzylpyrazolopyrimidine triphosphate (3-benzyl-PPTP) proved to be a substrate for a mutant of the MAP kinase p38 (p38-T106G/A157L/L167A). 3-Benzyl-PPTP was preferred by v-Src (T338G) (k(cat)/K(M) = 3.2 x 10(6) min(-)(1) M(-)(1)) over ATP or the previously described ATP analogue, N(6) (benzyl) ATP. For the kinase CDK2 (F80G)/cyclin E, 3-benzyl-PPTP demonstrated catalytic efficiency (k(cat)/K(M) = 2.6 x 10(4) min(-)(1) M(-)(1)) comparable to ATP (k(cat)/K(M) = 5.0 x 10(4) min(-)(1) M(-)(1)) largely due to a significantly better K(M) (6.4 microM vs 530 microM). In kinase protein substrate labeling experiments both 3-benzyl-PPTP and 3-phenyl-PPTP prove to be over 4 times more orthogonal than N(6)-(benzyl)-ATP with respect to the wild-type kinases found in murine spleenocyte cell lysates. These experiments also demonstrate that [gamma-(32)P]-3-benzyl-PPTP is an excellent phosphodonor for labeling the direct protein substrates of CDK2 (F80G)/E in murine spleenocyte cell lysates, even while competing with cellular levels (4 mM) of unlabeled ATP. The fact that this new more highly orthogonal nucleotide is accepted by three widely divergent kinases studied here suggests that it is likely to be generalizable across the entire kinase superfamily.     

Synthesis and esterolytic activity of catalytic peptide dendrimers

Peptide dendrimers were prepared by solid-phase peptide synthesis. Monomeric dendrimers were first obtained by assembly of a hexapeptide sequence containing alternate standard alpha-amino acids with diamino acids as branching units. The monomeric dendrimers were then dimerized by disulfide-bridge formation at the core cysteine. The synthetic strategy is compatible with functional amino acids and different diamino acid branching units. Peptide dendrimers composed of the catalytic triad amino acids histidine, aspartate, and serine catalyzed the hydrolysis of N-methylquinolinium salts when the histidine residues were placed at the outermost position. The dendrimer-catalyzed hydrolysis of 7-isobutyryl-N-methylquinolinium followed saturation kinetics with a rate constant of catalysis/rate constant without catalysis (k(cat)/k(uncat)) value of 3350 and a rate constant of catalysis/Michaelis constant (k(cat)/K(M)) value 350-fold larger than the second-order rate constant of the 4-methylimidazole-catalyzed reaction; this corresponds to a 40-fold rate enhancement per histidine side chain. Catalysis can be attributed to the presence of histidine residues at the surface of the dendrimers.     

Cross‐Linked Micelles with Enzyme‐Like Active Sites for Biomimetic Hydrolysis of Activated Esters

Enzymes have substrate‐tailored active sites with optimized molecular recognition and catalytic features. Although many different platforms have been used by chemists to construct enzyme mimics, it is challenging to tune the structure of their active sites systematically. By molecularly imprinting template molecules within doubly cross‐linked micelles, we created protein‐sized nanoparticles with catalytically functionalized binding sites. These enzyme mimics accelerated the hydrolysis of activated esters thousands of times over the background reaction, whereas the analogous catalytic group (a nucleophilic pyridyl derivative) was completely inactive in bulk solution under the same conditions. The template molecules directly controlled the size and shape of the active site and modulated the resulting catalyst's performance at different pHs. The synthetic catalysts displayed Michaelis–Menten enzymatic behavior and, interestingly, reversed the intrinsic reactivity of the activated esters during the hydrolysis.     

Rapid hydrolysis of phosphate ester promoted by Ce(IV) conjugating with a β-cyclodextrin monomer and dimer

Two N-donor ligands (L(1) and L(2)) derived from a β-cyclodextrin (βCD) monomer and dimer were employed to mediate the hydrolytic activity and stability of the Ce(IV) ion in aqueous solution. Complexes Ce(IV)-L(1) and Ce(IV)-L(2) were prepared in situ and characterized by means of UV-vis and NMR measurements. Ce(IV)-L(1) catalyzed the hydrolysis of a DNA model, bis(4-nitrophenyl)phosphate (BNPP) with k(cat) = 5.2 × 10(-3) s(-1) (half-life t(1/2) ≈ 2 minutes) under mild conditions, which represented an approximate 130 million-fold acceleration with respect to the spontaneous hydrolysis of BNPP. The dinuclear species, [Ce(2)L(1)(2)(OH)(5)](3+), contributed splendidly to the catalytic efficiency which echoed the active species postulation of [Ce(2)(OH)(7)](+) in the literature. Ce(IV)-L(2) exhibited efficient binding with BNPP giving 1/K(M) = 2.1 × 10(5) M(-1) which exceeded other Ce(IV) species, e.g. [Ce(4)(OH)(15)](+), by 2 orders of magnitude, which highlighted the hydrophobicity effect of βCDs. Such a highly binding affinity leads to the second-order rate constant, k(cat)/K(M) = 2.3 × 10(2) M(-1) s(-1), which probably ranks as the highest in the non-enzymatic cleavage of BNPP under similar conditions. Additionally, Ce(IV)-L(2) showed favorable tolerance to basic aqua owing to the bulky protection of double βCD pendants.     

Mechanistic studies of the isomerization of peroxynitrite to nitrate catalyzed by distal histidine metmyoglobin mutants

Hemoproteins are known to react with the strong nitrating and oxidizing agent peroxynitrite according to different mechanisms. In this article, we show that the iron(iii) forms of the sperm whale myoglobin (sw Mb) mutants H64A, H64D, H64L, F43W/H64L, and H64Y/H93G catalyze the isomerization of peroxynitrite to nitrate. The two most efficient catalysts are H64A (k(cat) = (5.8 +/- 0.1) x 10(6) M(-1) s(-1), at pH 7.5 and 20 degrees C) and H64D metMb (k(cat) = (4.8 +/- 0.1) x 10(6) M(-1) s(-1), at pH 7.5 and 20 degrees C). The pH dependence of the values of k(cat) shows that HOONO is the species which reacts with the heme. In the presence of physiologically relevant concentrations of CO(2) (1.2 mM), the decay of peroxynitrite is accelerated by these metMb mutants via the concurring reaction of HOONO with their iron(iii) centers. Studies in the presence of free added tyrosine show that the metMb mutants prevent peroxynitrite-mediated nitration. The efficiency of the different sw metMb mutants correlates with the value of k(cat). Finally, we show that sw WT-metMb is nitrated to a larger extent than horse heart metMb, a result that suggests that the additional Tyr151 is a site of preferential nitration. Again, the extent of nitration of the tyrosine residues of the metMb mutants correlates with the values of k(cat).     

Kinetics of N-glutaryl-L-phenylalanine p-nitroanilide hydrolysis catalyzed by alpha-chymotrypsin in aqueous solutions of dodecyltrimethylammonium bromide

The rate of hydrolysis of N-glutaryl-L-phenylalanine p-nitroanilide (GPNA) catalyzed by alpha-chymotrypsin (alpha-CT) has been measured in aqueous solutions of dodecyltrimethylammonium bromide (DTAB) at concentrations below and above the critical micelle concentration, as well as in the absence of surfactant. Under all the conditions employed, the reaction follows a Michaelis-Menten mechanism. The presence of the surfactant leads to superactivity below and above the critical micelle concentration (CMC), with a maximum reaction rate taking place near the CMC when the results are treated in terms of the analytical concentration of the substrate. A similar behavior was observed by working with the enzyme partially deactivated in the presence of 4 M urea. After correction to take into account the partitioning of the substrate between the micelles and the external media, the activity of the enzyme tends to remain almost constant above the corresponding CMCs. This results from a compensation of a decrease in the catalytic constant (k(cat)) and a decrease in the Michaelis constant (K(M)). The behavior of alpha-CT in the hydrolysis of GPNA in DTAB solutions is at variance with that previously reported for the hydrolysis of 2-naphthyl acetate in solutions of the same surfactant (E. Abuin, E. Lissi, R. Duarte, Langmuir 19 (2003) 5374). An explanation of the different effects of the surfactant on the behavior of the enzyme with both substrates is advanced, taking into account the complexity of the mechanism of the alpha-CT-mediated reaction, more specifically, in terms of different rate-limiting steps for the formation of the measured products.