Enolimine Tautomer Research Articles

PH studies on the mechanism of the pyridoxal phosphate-dependent dialkylglycine decarboxylase.

The pH dependence of the steady-state kinetic parameters for the dialkylglycine decarboxylase-catalyzed decarboxylation-dependent transamination between 2-aminoisobutyrate (AIB) and pyruvate is presented. The pH dependence of methylation and DTNB modification reactions, and spectroscopic properties, is used to augment the assignment of the kinetic pKa's to specific ionizations. The coincidence of pKa values (approximately 7.4) observed in kcat/KAIB, 1/KAIB, Kis for pyruvate, KPLP, and in absorbance and fluorescence titrations demonstrates that AIB is not a sticky substrate. It furthermore suggests that the decarboxylation step, or a conformational isomerization preceding it, limits the rate of the overall catalytic cycle. Coexisting, kinetically distinguishable conformers of DGD-PLP, originating from an alkali metal ion binding site, were previously demonstrated at pH 8.2 for DGD-PLP (Zhou, X., Toney, M. D. Biochemistry 37, 5761-5769). The pKa value of approximately 8.8 observed in kcat, kcat/KAIB, Kd for K+, spectrometric titrations, and the reaction of DGD-PLP with DTNB is tentatively assigned to the conformational change interconverting the two enzyme forms previously characterized. Three pKa's are observed in pH titrations of the DGD-PLP coenzyme absorbance. Individual spectra for the four ionization states are deconvoluted by fitting log-normal curves. All four ionization states have both ketoenamine and enolimine tautomers present. This and a review of spectral data in the literature lead to the conclusion that the pKa of approximately 7.4, which gives the largest spectral changes and controls kcat/KAIB, is not deprotonation of the aldimine nitrogen. Rather, it must be an active site residue whose ionization alters the ratio between ketoenamine and enolimine tautomers.

Time-resolved fluorescence of O-acetylserine sulfhydrylase catalytic intermediates.

The reaction of the substrate O-acetyl-L-serine (OAS) with the pyridoxal 5'-phosphate (PLP)-dependent enzyme O-acetylserine sulfhydrylase-A (OASS-A) proceeds via the transient formation of an external aldimine absorbing at 420 nm and a stable alpha-aminoacrylate intermediate absorbing at 330 and 465 nm. Stable external aldimine species are obtained by reaction of the enzyme with either the reaction product L-cysteine or the product analog L-serine. Static and time-resolved fluorescence emission properties of the coenzyme in the above catalytic intermediates have been used to directly probe the active site conformation at different stages of the catalytic pathway. Upon excitation at either 420 or 330 nm, the external aldimines with L-cysteine and L-serine exhibit a structured emission centered at 490 nm with a shoulder at 530 nm. Fluorescence decays upon excitation at 420 nm are best fitted using two components with lifetimes of 1.1 and 3.8 ns, with the fractional intensity of the slow component being 0.92 with L-cysteine and 0.75 with L-serine, respectively. The fast component, emitting at 530 nm, is attributed to a dipolar species formed in the excited state by proton dissociation, and the slow component, emitting at 490 nm, is attributed to a ketoenamine tautomer of the external aldimine. The slow component for external aldimine fluorescence decay is characterized by the same lifetime value as that of the internal aldimine with an increased fractional intensity, indicating that the distribution between the ketoenamine and the dipolar species is shifted toward the ketoenamine tautomer in the external aldimine, compared to the internal aldimine. Differences in equilibrium distribution of ketoenamine and enolimine tautomers can also account for differences in the emission properties of the external aldimines of L-cysteine and L-serine. The alpha-aminoacrylate species is characterized by a relatively weak emission. Upon excitation at 330 nm, the emission exhibits two bands centered at 420 and 540 nm, whereas upon excitation at 420 nm the emission bands are centered at 500 and 540 nm, and upon excitation at 465 nm, the main absorbance peak of the alpha-aminoacrylate species, the emission spectrum shows a band at 540 nm. The fluorescence decays, upon excitation at 330 nm, are best fitted using three components with lifetime values similar to those found for the internal aldimine, with the slow component predominating. Species-associated spectra, collected between 400 and 520 nm upon excitation at 350 nm, indicate the presence of a fast component overlapping the slow component on the blue side of the emission spectrum, as detected for the internal aldimine. When the excitation wavelength is 420 nm, there are only two components with the fast one predominating. A further increase in the fractional intensity of the fast component is observed upon excitation at 465 nm. The weak emission and the short lifetime of the emission excited at 465 nm indicate that this alpha-aminoacrylate tautomer interacts significantly with neighboring groups of the protein matrix and may be endowed with a higher mobility than the external aldimine.

Two Schiff Base Ligands Derived from 1,2-Diaminoethane

The structures of two Schiff base ligands, 2,2′-[(1,2-ethanediyl)bis(nitrilopropylidyne)]bisphenol, C20H24N2O2, (I) and 2,2′-{(1,2-ethanediyl)bis[nitrilo(phenyl)methylidyne]}bisphenol, C28H24N2O2, (II), are reported. The molecular structure of (I) is centrosymmetric and therefore has a trans conformation. The asymmetric unit for structure (II) contains two molecules, but both have gauche arrangements. Structures (I) and (II) both contain the enolimine tautomers and exhibit intramolecular hydrogen bonding.

Electroreduction of the Schiff base of pyridoxal-5′-phosphate and hexylamine in dimethylformamide and methanol. Effect of the self-protonation

Abstract The present paper reports an investigation of the electrochemical behaviour of the pyridoxal-5′-phosphate and hexylamine Schiff base in water, methanol and dimethylformamide using linear sweep voltammetry, absorption spectroscopy and controlled-potential electrolysis. The global reduction corresponds to the formation of a secondary amine, hexyl-pyridoxamine-5′-phosphate, by the uptake of two electrons and two protons in the electrode process. In the aprotic solvent, where the enolimine tautomer is favoured, evidence of a self-protonation effect on the electroreduction mechanism is obtained.

Nitrogen bridgehead compounds. Part 64. Protonation of 9-formyltetrahydropyrido[1,2-a]pyrimidin-4-ones and their analogues

As shown by 1H, 13C, and 15N n.m.r. spectroscopy, protonation of 9-formyltetrahydropyrido[1,2-a] pyrimidin-4-ones takes place at the formyl oxygen atom. As a result, the enamine tautomer, predominant in the base form, is transformed into a protonated enolimine tautomer having an exocyclic double bond. 15N N.m.r. shifts reflect not only the state of the tautomeric equilibria but also the state of Z/E isomerism and the effect of remote substituents.

Studies of 6-fluoropyridoxal and 6-fluoropyridoxamine 5'-phosphates in cytosolic aspartate aminotransferase.

The chemical and spectroscopic properties of 6-fluoropyridoxal 5'-phosphate, of its Schiff base with valine, and of 6-fluoropyridoxamine 5'-phosphate have been investigated. The modified coenzymes have also been combined with the apo form of cytosolic aspartate aminotransferase, and the properties of the resulting enzymes and of their complexes with substrates and inhibitors have been recorded. Although the presence of the 6-fluoro substituent reduces the basicity of the ring nitrogen over 10 000-fold, the modified coenzymes bind predominately in their dipolar ionic ring forms as do the natural coenzymes. Enzyme containing the modified coenzymes binds substrates and dicarboxylate inhibitors normally and has about 42% of the catalytic activity of the native enzyme. The fluorine nucleus provides a convenient NMR probe that is sensitive to changes in the state of protonation of both the ring nitrogen and the imine or the -OH group of free enzyme and of complexes with substrates or inhibitors. The NMR measurements show that the ring nitrogen of bound 6-fluoropyridoxamine phosphate is protonated at pH 7 or below but becomes deprotonated at high pH around a pKa of 8.2. The bound 6-fluoropyridoxal phosphate, which exists as a Schiff base with a dipolar ionic ring at high pH, becomes protonated with a pKa of approximately 7.1, corresponding to the pKa of approximately 6.4 in the native enzyme. Below this pKa a single 19F resonance is seen, but there are two light absorption bands corresponding to ketoenamine and enolimine tautomers of the Schiff base. The tautomeric ratio is altered markedly upon binding of dicarboxylate inhibitors. From the chemical shift values, we conclude that during the rapid tautomerization a proton is synchronously moved from the ring nitrogen (in the ketoenamine) onto the aspartate-222 carboxylate (in the enolimine). The possible implications for catalysis are discussed.

The effect of temperature and the kinetics of the hydrolysis of some β-diketone Schiff bases

The first order rate constants ( k obsd.) of the hydrolysis of Schiff bases derived from β-diketones were measured at different temperatures and pH in phosphate and acetate buffers. The results show that some molecules hydrolyze more slowly due to the existence of a high proportion of a hydrogen bonded enolimine tautomer, whereas the rates of hydrolysis of others are accelerated due to the formation of a species which has not been suggested previously containing two positive charge centres adjacent to each other. This species allows the rapid formation of the carbinolamine, the well-known intermediate during the hydrolysis of Schiff bases. From the effect of temperature, the activation parameters of the hydrolysis have been calculated although definite trends have not been established due to the random variation of their values with the nature of the substituent.

Spectrophotometric titrations of pyridoxal Schiff's bases in methanol using glass electrode for control of acidity

Schiff's bases prepared by mixing pyridoxal, pyridoxal 5′-phosphate (PLP), or salicylaldehyde (SA) with amino compounds, having a different structure at carbon-1, were spectrophotometrically titrated in methanol by using glass electrode and operational pH ∗ scale for control of the acidity. The titrations were carried out with methanolic HCl from alkaline solutions, obtained by adding sodium methoxide. The solutions were titrated so rapidly that the acetal forms of the Schiff's bases did not appear in considerable amounts. When the Schiff's bases were prepared from 2-amino acids, the p K ∗ values of phenolic hydroxyls were higher than those of the Schiff's bases made from 3- or 4-amino acids or from alkylamines. Carboxylate of the 2-amino acid also increased the relative amount of the ketoenamine tautomer at pH ∗ 6–10, as compared to the other amino compounds studied. The effect of carboxylate on the tautomerism was interpreted to be due to internal hydrogen bonding between carboxylate anion, azomethine nitrogen and phenolic hydroxyl. In addition, phosphate of pyridoxal 5′-phosphate was deduced to force carboxylate, by ionic repulsion, to a favorable conformation for the internal hydrogen bonding. The protonation of carboxylate of 2-amino acid imines (p K ∗ about 5) brought about a great change toward enolimine tautomer due to a rupture of the internal hydrogen bonding. Below p K ∗ of about 3, pyridine nitrogen protonized and concomitantly the tautomeric equilibrium reshifted on ketoenamine. With salicylaldehyde imines an immonium ion appeared at about the same pH ∗. The present results are applicable to the predicting of spectral changes in hydrophobic active sites of pyridoxal 5′-phosphate enzymes.

ChemInform Abstract: MODEL STUDIES OF PYRIDOXAL SCHIFF′S BASES. COPLANARITY AND INTRAMOLECULAR HYDROGEN BONDING

The interactions between the pi cloud of the aromatic ring and the pi-electron pair of the imine double bond of aromatic oximes as model compounds of pyridoxal Schiff's bases have been studied by high-resolution carbon-13 magnetic resonance spectroscopy. The coplanarity and intramolecular hydrogen bonding have been determined by 13C-1H long range couplings. This detailed investigation of 13C-1H coupling also provides unambiguous proof of the existence of the enol-imine tautomers in chloroform and dimethyl sulfoxide solutions. The tautomerism between the enol-imine and keto-enamine is discussed.

Tautomerism and hydrogen bonding in bilirubin and biliverdin

1. 1. Infrared and NMR spectrometric studies on bilirubin and biliverdin and their derivatives suggest that whereas bilirubin occurs as the enol-imine tautomer, biliverdin and its dimethyl ester are the lactam form. 2. 2. Methylation of bilirubin results in a shift in the absorption maximum from 450 mμ to 405 mμ due to assumption of the lactam form. The change is caused by loss of hydrogen bonding between the propionic acid side chains which hold the molecule in a conformation suitable for hydrogen bonding between the two terminal hydroxyl groups. 3. 3. The failure of bilirubin to form stable zinc complexes and its weakly basic character are discussed in terms of the data leading to the conclusions above.