Solvent Kinetic Isotope Research Articles

Isotope effects in the enzymatic oxidation of tryptamine to 3-indolyl-acetaldehyde

The reaction mechanisms of the enzymatic deamination of tryptamine catalysed by the enzyme monoamine oxidase (MAO, EC 1.4.3.4) were investigated using the kinetic isotope effect and solvent isotope effect methods. The numerical values of these deuterium effects in the (1S) and (1R) positions of tryptamine were determined using the non-competitive spectrophotometry. The deuterium-labelled isotopologue [(1S)-2H]tryptamine was obtained in two steps by enzymatic coupling of indole with S-methyl-l-cysteine in a deuterated medium followed by enzymatic decarboxylation of the resulting [2-2H]-l-tryptophan. [(1R)-2H]tryptamine was obtained by enzymatic decarboxylation of l-tryptophan in the fully deuterated medium.

Kinetic Isotope Effects Support the Twisted Amide Mechanism of Pin1 Peptidyl-Prolyl Isomerase

The Pin1 peptidyl-prolyl isomerase catalyzes isomerization of pSer/pThr-Pro motifs in regulating the cell cycle. Peptide substrates, Ac-Phe-Phe-phosphoSer-Pro-Arg-p-nitroaniline, were synthesized in unlabeled form, and with deuterium-labeled Ser-d3 and Pro-d7 amino acids. Kinetic data were collected as a function of Pin1 concentration to measure kinetic isotope effects (KIEs) on catalytic efficiency (kcat/Km). The normal secondary (2°) KIE value measured for the Ser-d3 substrate (kH/kD = 1.6 ± 0.2) indicates that the serine carbonyl does not rehybridize from sp(2) to sp(3) in the rate-determining step, ruling out a nucleophilic addition mechanism. The normal 2° KIE can be explained by hyperconjugation between Ser α-C-H/D and C═O and release of steric strain upon rotation of the amide bond from cis to syn-exo. The inverse 2° KIE value (kH/kD = 0.86 ± 0.08) measured for the Pro-d7 substrate indicates rehybridization of the prolyl nitrogen from sp(2) to sp(3) during the rate-limiting step of isomerization. No solvent kinetic isotope was measured by NMR exchange spectroscopy (kH2O/kD2O = 0.92 ± 0.12), indicating little or no involvement of exchangeable protons in the mechanism. These results support the formation of a simple twisted amide transition state as the mechanism for peptidyl prolyl isomerization catalyzed by Pin1. A model of the reaction mechanism is presented using crystal structures of Pin1 with ground state analogues and an inhibitor that resembles a twisted amide transition state.

A quantitative analysis of spontaneous isoaspartate formation from N-terminal asparaginyl and aspartyl residues

The formation of isoaspartate (isoAsp) from asparaginyl or aspartyl residues is a spontaneous post-translational modification of peptides and proteins. Due to isopeptide bond formation, the structure and possibly function of peptides and proteins is altered. IsoAsp modifications within the peptide chain have been reported for many cytosolic proteins. Amyloid peptides (Aβ) deposited in Alzheimer's disease may carry an N-terminal isoAsp-modification. Here, we describe a quantitative investigation of isoAsp-formation from N-terminal Asn and Asp using model peptides similar to the Aβ N-terminus. The study is based on a newly developed separation of peptides using capillary electrophoresis (CE). 1H NMR was employed to validate the basic finding of N-terminal isoAsp-formation from Asp and Asn. Thereby, the isomerization of Asn at neutral pH (0.6 day(-1), peptide NGEF) is approximately six times faster than that within the peptide chain (AANGEF). The difference in velocity between Asn and Asp isomerization is approximately 50-fold. In contrast to N-terminal Asn, Asp isomerization is significantly accelerated at acidic pH. The kinetic solvent isotope (kD2O/kH2O) effect of 2.46 suggests a rate-limiting proton transfer in isoAsp-formation. The proton inventory is consistent with transfer of one proton in the transition state, supporting the previous notion of rate-limiting deprotonation of the peptide backbone amide during succinimide-intermediate formation. The study provides evidence for a spontaneous N-terminal isoAsp-formation within peptides and might explain the accumulation of N-terminal isoAsp in amyloid deposits.

Kinetic and solvent deuterium isotope effects in the oxidation of putrescine catalysed by enzyme diamine oxidase

In this study, the kinetic isotope effects and solvent isotope effects in the reaction of the deamination of [(1R)-2H ] putrescine – catalysed by enzyme diamine oxidase (EC 1.4.3.6) – were determined using a non-competitive spectroscopic method. Putrescine, stereospecifically labelled with deuterium, was obtained by enzymatic decarboxylation of l-ornithine that was carried out in a fully deuteriated incubation medium.

Kinetic and Chemical Mechanisms of Homocitrate Synthase from Thermus thermophilus

The homocitrate synthase from Thermus thermophilus (TtHCS) is a metal-activated enzyme with either Mg(2+) or Mn(2+) capable of serving as the divalent cation. The enzyme exhibits a sequential kinetic mechanism. The mechanism is steady state ordered with α-ketoglutarate (α-Kg) binding prior to acetyl-CoA (AcCoA) with Mn(2+), whereas it is steady state random with Mg(2+), suggesting a difference in the competence of the E·Mn·α-Kg·AcCoA and E·Mg·α-Kg·AcCoA complexes. The mechanism is supported by product and dead-end inhibition studies. The primary isotope effect obtained with deuterioacetylCoA (AcCoA-d(3)) in the presence of Mg(2+) is unity (value 1.0) at low concentrations of AcCoA, whereas it is 2 at high concentrations of AcCoA. Data suggest the presence of a slow conformational change induced by binding of AcCoA that accompanies deprotonation of the methyl group of AcCoA. The solvent kinetic deuterium isotope effect is also unity at low AcCoA, but is 1.7 at high AcCoA, consistent with the proposed slow conformational change. The maximum rate is pH independent with either Mg(2+) or Mn(2+) as the divalent metal ion, whereas V/K(α-Kg) (with Mn(2+)) decreases at low and high pH giving pK values of about 6.5 and 8.0. Lysine is a competitive inhibitor that binds to the active site of TtHCS, and shares some of the same binding determinants as α-Kg. Lysine binding exhibits negative cooperativity, indicating cross-talk between the two monomers of the TtHCS dimer. Data are discussed in terms of the overall mechanism of TtHCS.

Hydrolysis of N-Phenylalkanesulfinamides in Aqueous Mineral Acids

The acid-catalyzed hydrolysis of N-phenylalkanesulfinamides (RSONHPh; 1, R = iPr; 2, R = tBu; 3, R = 1-adamantyl) has been studied in aqueous mineral acids. Hydrolysis was found to proceed via a slow spontaneous (uncatalyzed) pathway, an A-2 (bimolecular) acid-catalysis pathway, and an acid-dependent nucleophilic catalysis pathway, the last of which predominates in hydrobromic and hydrochloric acid solutions. A mechanistic switch over from A-2 to A-1 was detected for compounds 2 and 3 in concentrated sulfuric acid. Order of catalytic activity, effect of added salts, Arrhenius parameters, kinetic solvent isotope, and solvent effects are all consistent with the proposed mechanisms.GRAPHICAL ABSTRACT

ChemInform Abstract: Reactions of Carbonyl Compounds in Basic Solutions. Part 15. The Alkaline Hydrolysis of N-Methyl, N-Phenyl and Bicyclo Lactams, Penicillins and N-Alkyl-N-methylacetamides

The rate coefficients for the alkaline hydrolysis of a series of N-methyl, N-phenyl and bicyclo lactams, penicillins and N-alkyl-N-methylacetamides in water or in aqueous dimethyl sulphoxide have been measured at several temperatures. The reactions were first order in both substrates and base. The reactivities are related to the structures of the substrates, especially N-substitution, ring size and fused ring effects. The reactivities, as well as the activation parameters, kinetic solvent and solvent isotope effects, are used to suggest the detailed mechanisms. All the β-lactams appear to react with rate-determining addition of hydroxide anion; while the N-alkyl γ- and δ-lactams and N-alkyl-N-methylacetamides have rate-determining ring fission of the tetrahedral adduct, assisted by water-catalysis. The reactivity of penicillin in alkaline hydrolysis has been analysed by the studies of model compounds. These show that the increased reactivity of the penicillin β-lactam ring arises from the direct action of the fused ring structure and the acylamino side-chain.

Reduction of O2 to Superoxide Anion (O2•-) in Water by Heteropolytungstate Cluster-Anions

Fundamental information concerning the mechanism of electron transfer from reduced heteropolytungstates (POM(red)) to O2, and the effect of donor-ion charge on reduction of O2 to superoxide anion (O2.-), is obtained using an isostructural series of 1e--reduced donors: alpha-X(n+)W12O40(9-n)-, X(n+) = Al3+, Si4+, P5+. For all three, a single rate expression is observed: -d[POM(red)]/dt = 2k12[POM(red)][O2], where k12 is for the rate-limiting electron transfer from POM(red) to O2. At pH 2 (175 mM ionic strength), k12 increases from 1.4 +/- 0.2 to 8.5 +/- 1 to 24 +/- 2 M-1s-1 as Xn+ is varied from P5+ (3red) to Si4+ (2red) to Al3+ (1red). Variable-pH data (for 1red) and solvent-kinetic isotope (KIE = kH/kD) data (all three ions) indicate that protonated superoxide (HO2.) is formed in two steps--electron transfer, followed by proton transfer (ET-PT mechanism--rather than via simultaneous proton-coupled electron transfer (PCET). Support for an outersphere mechanism is provided by agreement between experimental k12 values and those calculated using the Marcus cross relation. Further evidence is provided by the small variation in k12 observed when Xn+ is changed from P5+ to Si4+ to Al3+, and the driving force for formation of O2.- (aq), which increases as cluster-anion charge becomes more negative, increases by nearly +0.4 V (a decrease of >9 kcal mol-1 in DeltaG degrees ). The weak dependence of k12 on POM reduction potentials reflects the outersphere ET-PT mechanism: as the anions become more negatively charged, the "successor-complex" ion pairs are subject to larger anion-anion repulsions, in the order [(3(ox)3-)(O2.-)]4- < [(2(ox)4-)(O2.-)]5- < [(1(ox)5-)(O2.-)]6-. This reveals an inherent limitation to the use of heteropolytungstate charge and reduction potential to control rates of electron transfer to O2 under turnover conditions in catalysis.

Reactions of carbonyl compounds in basic solutions. Part 25.1 The mechanism of the base-catalysed ring fission of 3,4-diphenylcyclobut-3-ene-1,2-diones

The rate coefficients for the base-catalysed ring fission of a series of substituted 3,4-diphenylcyclobut-3-ene-1,2-diones to give the corresponding (Z)-2-oxo-3,4-diphenylbut-3-enoic acids have been determined in 50% (v/v) aqueous dimethyl sulfoxide (DMSO) at 25.0 and 45.0 °C, as well as for a limited series in 70% (v/v) aqueous DMSO. The activation parameters have been calculated. The effect of both mono- and di-substitution of the phenyl groups has been studied and gives a Hammett ρ value of ca. 1.3 in 50% aqueous DMSO at 25.0 °C. The kinetic solvent isotope and solvent effect and enrichment in 18O-enriched water have been studied. The product composition of the reaction of the monosubstituted compounds gave a ρ value of ca. 0.9. All the evidence indicates a mechanistic pathway which proceeds by a rapid, reversible addition of hydroxide anion to the dione, followed by a benzilic acid-type rearrangement to form a 1-hydroxycyclopropene-1-carboxylic acid. The latter then suffers ring fission to give the final product.

The Hydrolysis of Phthalide, Thiophthalide, and Methyl o-Methoxybenzoate in Highly Alkaline Media. Curvature in the khyd vs [OH-] Profile

The hydrolyses of phthalide (2a) and thiophthalide (3) have been stu- died in highly alkaline media, T=25 o C, μ=3 (HCl). For both esters, an upward curvature in the plot of k hyd vs [OH - ] is observed, sugges- tive of the onset of a second order in [OH - ] process in the hydrolysis. Carbonyl 18 O-exchange studies indicated that no 18 O is lost from the ester recovered from the hydrolysis media at times up to 3t 1/2 hydrolysis. Solvent kinetic isotope (skie) studies indicate that for 3, (k hyd ) H2O/D2O is inverse throughout the entire range of [OL - ]

Isotope effects on the mechanism of calcineurin catalysis: kinetic solvent isotope and isotope exchange studies

The reaction scheme of calcineurin was examined with kinetic and physical approaches. Proton inventory studies of the calcineurin-catalyzed hydrolysis of para-nitrophenyl phosphate were done to probe the role of proton transfer in the mechanism. Control experiments determined that the solvent did not cause the irreversible inactivation of the enzyme and had no effect on the dependence on metal ion or calmodulin. A solvent isotope effect was observed on the V max K m term, but not the V max term. The isotope effect was modest with a value of 1.35. Proton inventory data could be fit by multiple parameter sets. The parameter sets yielded fractionation factors of 0.73 for a one-proton transfer or 0.85 for a two-proton transfer. These values compare to the value of 0.69 for reactions involving a water molecule or hydroxide coordinated to metal ion. A chemical mechanism consistent with the proton inventory data and other information about calcineurin catalysis is presented. The simplest model for catalysis involves a single proton transfer from water coordinated to metal that is reasoned to occur during association of the substrate with calcineurin. Questions about the reaction intermediate were also addressed. Attempts to monitor a phosphate-water exchange reaction with 31P nuclear magnetic resonance spectroscopy were unsuccessful. Failure to observe an exchange reaction suggests that no phosphoryl enzyme is formed during the progress of the reaction. Together these data are explained by a model in which cleavage of the phosphate ester bond is catalyzed by a water (hydroxide) molecule coordinated to a divalent metal ion without the formation of a covalent intermediate.

Kinetics and mechanism of the denitrosation of nitrosamines in ethanol

Rate constants have been determined for the denitrosation of N-methyl-N-nitrosoaniline (NMNA), N-nitrosodiphenylamine (NDA), and N-methyl-N-nitrosotoluene-p-sulphonamide (MTS) in ethanolic solutions of hydrogen chloride. For both NMNA and NDA the change is reversible in the absence of a trap for the nitrosyl chloride formed; the equilibrium position is dependent upon [HCl]. A kinetic analysis in the case of NMNA gives the rate constants for both the forward and reverse reactions. A more accurate determination of the forward rate constant was obtained for both NMNA and NDA by using an excess of added ascorbic acid to remove the free nitrosating species. Under these conditions the rate law, rate =k2[Nitrosamine][HCl], prevails. The reaction of MTS was irreversible even in the absence of a nitrite trap and the same rate law was established. All three reactants show the absence of catalysis by added sodium bromide and potassium thiocyanate and gave kinetic deuterium solvent isotope effects kEtOH/kEtOD in the range 2.6–3.8. The results are in accord with a rate-determining proton transfer to the nitrosamine, contrasting with the behaviour (of NMNA and NDA) in water solvent. The rate of denitrosation of NMNA decreases as water is added to the solvent, and nucleophilic catalysis begins to be effective. The Fischer–Hepp rearrangement of NDA is observed when ascorbic acid is absent; the rate constant for rearrangement is much less than that for denitrosation at the same acidity.

Kinetic deuterium solvent isotope effects in the pyridine-catalysed iodination of 2-nitropropane in aqueous and methanolic solutions

The rate of proton transfer from 2-nitropropane to pyridine was measured in the solvents H2O, D2O, CH3OH, and CH3OD by the observation of the rate of disappearance of added iodine at 25 °C. The reaction is ca. 150 times slower in methanol than in water, but the factor by which the rate constant is changed when hydroxylic protium is replaced by deuterium is slight and practically the same in both media (kH/kDca. 1.05 ± 0.02). The result rules out from further consideration the idea that the marked difference in solvent isotope effects for the corresponding reactions of the same substrate with lyate ions in these two solvents is due to some general and unexplained solvent effect rather than to an effect specifically associated with the chemical differences between lyate ions in water and alcoholic media.

Intramolecular catalysis. Part 2. Mechanism of hydrolysis of alkyl 8-hydroxy-1-naphthoates

The hydrolysis of alkyl 8-hydroxy-1 -naphthoates in alkaline 30%(v/v) dioxan–water has been investigated. The pH′-rate profiles, activation parameters, comparison with the rates of hydrolysis of model compounds and 8-hydroxy1 -naphthoic acid lactone, and kinetic solvent isotope and other effects indicate a mechanism involving intramolecular catalysis, either by general base or by general acid specific base. The significance of the results is discussed in relation to the factors governing such mechanistic pathways.

Reactions in strongly basic media. Part V. The kinetics and mechanism of the alkaline hydrolysis of substituted 2-alkoxytropones

The rate coefficients for the alkaline hydrolysis of a series of substituted 2-alkoxytropones in 40% v/v aqueous dioxan and 40% v/v aqueous dimethyl sulphoxide have been measured at 30·0°. The effect of variation of the composition of the medium and the temperature has been investigated for the hydrolysis of selected substrates. Kinetic solvent isotope and salt effects have also been studied. These measurements have also been carried out on two model systems, 3- and 4-substituted methyl benzoates and 4-substituted 1-methoxy-2-nitrobenzenes. The effects of substitution in these three systems have been assessed by linear free energy relations. The alkaline hydrolysis of the 2-alkoxytropones appears to proceed by an addition–elimination mechanism, involving a rate-determining direct attack by hydroxide anion at the 2-position.