Jacek Wierzchowski

Properties of two unusual, and fluorescent, substrates of purine-nucleoside phosphorylase: 7-methylguanosine and 7-methylinosine

The properties of two unusual substrates of calf spleen purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1), 7-methylguanosine and 7-methylinosine, are described. The corresponding bases, 7-methylguanine and 7-methylhypoxanthine, are neither substrates in the reverse, synthetic reaction, nor inhibitors of the phosphorolysis reaction. Both nucleosides exhibit fluorescence, which disappears on cleavage of the glycosidic bond, providing a new convenient procedure for continuous fluorimetric assay of enzymatic activity. For 7-methylguanosine at neutral pH and 25 degrees C, Vmax = 3.3 mumol/min per unit enzyme and Km = 14.7 microM, so that Vmax/Km = 22 X 10(-2)/min per unit as compared to 8 X 10(-2) for the commonly used substrate inosine. The permissible initial substrate concentration range is 5-100 microM. Enzyme activity may also be monitored spectrophotometrically. For 7-methylinosine, Vmax/Km is much lower, 2.4 X 10(-2), but its 10-fold higher fluorescence partially compensates for this, and permits the use of initial substrate concentrations in the range 1-500 microM. At neutral pH both substrates are mixtures of cationic and zwitterionic forms. Measurements of pH-dependence of kinetic constants indicated that the cationic forms are the preferred substrates, whereas the monoanion of inosine appears to be almost as good a substrate as the neutral form. With 7-methylguanosine as substrate, and monitoring of activity fluorimetrically and spectrophotometrically, inhibition constants were measured for several known inhibitors, and the results compared with those obtained with inosine as substrate, and with results reported for the enzyme from other sources.

Fluorescence emission properties of 8-azapurines and their nucleosides, and application to the kinetics of the reverse synthetic reaction of purine nucleoside phosphorylase

An extensive study has been made of the fluorescence emission properties of the neutral and ionic forms in aqueous medium of the azapurine nucleosides, 8-azaadenosine (8-azaAdo), 8-azainosine (8-azaIno), 8-azaguanosine (8-azaGuo), and their aglycons. The fluorescence of 8-azaGuo at pH 7 originates from its anionic species (pKa = 8.05, phi= 0.55), as is also the case for 8-azaIno (pKa = 8.0, phi = 0.02), whereas 8-azaAdo is a strong emitter (phi = 0.06) as the neutral species. By contrast the corresponding free 8-azapurines are only weakly fluorescent in aqueous medium, with the exception of 8-azaguanine (8-azaG). Examination of the emission properties of N-substituted 8-azaguanines demonstrated that the observed blue emission of the neutral form of 8-azaG (phi = 0.05 to 0.33, dependent on lambda exc) originates from a minor tautomer of the compound, the N(8)-H form, present to the extent of 10-15%; while the principal N(9)-H tautomer is virtually nonfluorescent. The 8-azapurines are substrates of purine nucleoside phosphorylase (PNP), leading to their irreversible conversion to the corresponding nucleosides in the synthetic pathway of this enzyme. The fluorescent properties of these compounds, together with spectrophotometric methods, were applied to determine the basic kinetic parameters for synthesis of 8-azapurine nucleosides by PNP from mammalian (calf spleen) and bacterial (Escherichia coli) sources. The fluorimetric method was also used to determine the kinetic parameters for the second substrate, alpha-D-ribose 1-phosphate, and for the analytical titration of the latter in solution. The pH optimum of the reverse synthetic PNP reaction with 8-azapurines as substrates is below pH 7, due to their enhanced acidity in comparison with natural purines. The 8-azapurine nucleosides, but not their aglycons, are reasonably good inhibitors of phosphorolysis of Ino and Guo by E. coli PNP. The most effective is 8-azaIno (Ki approximately 20 microM), also the only one to inhibit phosphorolysis by the calf spleen enzyme (Ki approximately 40 microM). The nature of this inhibition is apparently uncompetitive.

Fluorometric assays for isozymes of human alcohol dehydrogenase

Two new fluorogenic substrates for human alcohol dehydrogenase (ADH), 4-methoxy-1-naphthaldehyde (IA) and 6-methoxy-2-napthaldehyde (IIA), are described. The 2-naphthaldehyde derivative fluoresces in aqueous media with a quantum yield of 0.22 with an emission maximum at 450 nm, but the 1-naphthaldehyde shows only weak fluorescence. The corresponding alcohol reduction products, 4-methoxy-1-naphthalenemethanol (IB) and 6-methoxy-2-naphthalenemethanol (IIB), exhibit fluorescence in the near uv region with quantum yields of 0.36 and 0.26, respectively. The Km values for the individual homogenous class I ADH isozymes, with the above naphthaldehydes as substrates, range from 0.35 to 11.5 microM. The kappa cat values range from 70 to 610 min-1 and are thus comparable to those for the best ADH substrates. Except for the beta 1 beta 1 isozyme, IA is the preferred substrate for class I ADH isozymes while IIA is rapidly reduced by class II (pi-ADH). The sensitivity and specificity of the enzymatic assay with IA as substrate are demonstrated and provide the basis for the determination of class I ADH activity in human serum.

LUMINESCENCE STUDIES ON FORMYCIN, ITS AGLYCONE, AND THEIR N-METHYL DERIVATIVES: TAUTOMERISM, SITES OF PROTONATION AND PHOTOTAUTOMERISM

Abstract— A detailed study has been made of the luminescence spectra of 3‐β‐d‐ribofuranosyl‐7‐amino‐pyrazolo(4,3‐d)pyrimidine (formycin A), 3‐propyl‐7‐aminopyrazolo(4,3‐d)pyrimidine (7APP), and their various N‐methyl derivatives, at room temperature and in methanol‐water glasses at 77 K. Comparisons of the foregoing, together with the observed dependence of the emission spectra of formycin and 7APP on excitation wavelength, demonstrated that these consist of two tautomeric species, N(1)H and N(2)H, both of which emit at 300 and 77 K. The two tautomers may be distinguished by the location of the emission maxima, especially for phosphorescence, and quantum yields for emission.

Formycin A and its N-methyl analogues, specific inhibitors of E. coli purine nucleoside phosphorylase (PNP): induced tautomeric shifts on binding to enzyme, and enzyme→ligand fluorescence resonance energy transfer

Steady-state and time-resolved emission spectroscopy were used to study the interaction of Escherichia coli purine nucleoside phosphorylase (PNP) with its specific inhibitors, viz. formycin B (FB), and formycin A (FA) and its N-methylated analogues, N(1)-methylformycin A (m(1)FA), N(2)-methylformycin A (m(2)FA) and N(6)-methylformycin A (m(6)FA), in the absence and presence of phosphate (P(i)). Complex formation led to marked quenching of enzyme tyrosine intrinsic fluorescence, with concomitant increases in fluorescence of FA and m(6)FA, independently of the presence of P(i). Fluorescence of m(1)FA in the complex increased only in the presence of P(i), while the weak fluorescence of FB appeared unaffected, independently of P(i). Analysis of the emission, excitation and absorption spectra of enzyme-ligand mixtures pointed to fluorescence resonance energy transfer (FRET) from protein tyrosine residue(s) to FA and m(6)FA base moieties, as a major mechanism of protein fluorescence quenching. With the non-inhibitor m(2)FA, fluorescence emission and excitation spectra were purely additive. Effects of enzyme-FA, or enzyme-m(6)FA, interactions on nucleoside excitation and emission spectra revealed shifts in tautomeric equilibria of the bound ligands. With FA, which exists predominantly as the N(1)-H tautomer in solution, the proton N(1)-H is shifted to N(2), independently of the presence of P(i). Complex formation with m(6)FA in the absence of P(i) led to a shift of the amino-imino equilibrium in favor of the imino species, and increased fluorescence at 350 nm; by contrast, in the presence of P(i), the equilibrium was shifted in favor of the amino species, accompanied by higher fluorescence at 430 nm, and a higher affinity for the enzyme, with a dissociation constant K(d)=0.5+/-0.1 microM, two orders of magnitude lower than that for m(6)FA in the absence of P(i) (K(d)=46+/-5 microM). The latter was confirmed by analysis of quenching of enzyme fluorescence according to a modified Stern-Volmer model. Fractional accessibility values (f(a)) varied from 0.31 for m(1)FA to 0.70 for FA, with negative cooperative binding of m(1)FA and FB, and non-cooperative binding of FA and m(6)FA. For all nucleoside ligands, the best model describing binding stoichiometry was one ligand per native enzyme hexamer. Fluorescence decays of PNP, FA and their mixtures were best fitted to a sum of two exponential terms, with average lifetimes () affected by their interactions. Complex formation resulted in a 2-fold increase in of FA, and a 2-fold decrease in of enzyme fluorescence. The amplitude of the long-lifetime component also increased, confirming the shift of the tautomeric equilibrium in favor of the N(2)-H species. The findings have been examined in relation to enzyme-nucleoside binding deduced from structural studies.

Continuous fluorimetric assay of nucleotide pyrophosphatase. Kinetics, inhibitors, and extension to dinucleoside oligophosphatases

Abstract A simple and convenient procedure is described for the continuous fluorimetric assay of nucleotide pyrophosphatase (dinucleotide nucleotidohydrolase, EC 3.6.1.9) activity. It is based on the multifold increase in fluorescence emission intensity accompanying hydrolysis of a new fluorogenic substrate, ϵAp 2 ϵA (where ϵ = 1, N 6 -etheno), or of commercially available ϵNAD + and FAD, using potato nucleotide pyrophosphatase. The procedure is applicable to enzyme activity in tissue extracts, as well as to kinetic studies and screening of potential inhibitors. K m and V max / K m for the substrates, and K i values for various compounds, were evaluated. The most effective inhibitor of ϵAp 2 ϵA hydrolysis was the alternative substrate 8-bromo-NAD + ( K i = 7 μ M). The method is also useful for continuous assay of snake venom phosphodiesterase I-5′-exo-nuclease (EC 3.1.4.1). The preparation of ϵAp 2 ϵA is described, and spectral properties of the fluorogenic substrates listed. Both ϵAp 3 ϵA and ϵAp 4 ϵA were also shown to be fluorogenic substrates for nucleotide pyrophosphatase and phosphodiesterase I, and may be used for continuous fluorimetric assay of specific dinucleoside oligophosphatases.

Salivary Aldehyde Dehydrogenase: Activity towards Aromatic Aldehydes and Comparison with Recombinant ALDH3A1

A series of aromatic aldehydes was examined as substrates for salivary aldehyde dehydrogenase (sALDH) and the recombinant ALDH3A1. Para-substituted benzaldehydes, cinnamic aldehyde and 2-naphthaldehydes were found to be excellent substrates, and kinetic parameters for both salivary and recombinant ALDH were nearly identical. It was demonstrated that for the fluorogenic naphthaldehydes the only produced reaction product after incubation in saliva is the carboxylate.

Aromatic aldehydes as fluorogenic indicators for human aldehyde dehydrogenases and oxidases: substrate and isozyme specificity

Substrate properties of a number of potentially fluorogenic aromatic aldehydes of naphthalenes, phenanthrenes and anthracenes and of some coumarin aldehydes towards various forms of the human and rat aldehyde oxidase and dehydrogenase were examined using absorption and emission spectroscopy. It was demonstrated that recombinant human class 1 aldehyde dehydrogenase (ALDH-1) readily oxidizes naphthalene (except for those ortho-substituted), phenanthrene and coumarin aldehydes, whereas the class 3 enzyme (ALDH-3) from human saliva is active only towards 2-naphthaldehyde derivatives. The observed reaction rates in both cases are comparable to those of the best known substrates, and the Km values are typically in the sub-micromolar range. Aldehyde oxidases (AlOx), which are present in mammalian liver, reveal much broader substrate specificity, oxidizing nearly all the compounds examined, including those of the anthracene series, with maximum activity in the micromolar range of substrate concentration. In rat liver, nearly all AlOx activity was located in the cytosolic fraction.

Interactions of calf spleen purine nucleoside phosphorylase with 8-azaguanine, and a bisubstrate analogue inhibitor: implications for the reaction mechanism.

Abstract Interactions of calf spleen purine nucleoside phosphorylase (PNP) with a non-typical substrate, 8-azaguanine (8-azaG), and a bisubstrate analogue inhibitor, 9-(2-phosphonylmethoxyethyl)- 8-azaguanine (PME-azaG), were investigated by means of steady-state fluorescence spectroscopy. Both 8-azaG and PME-azaG form fluorescent complexes with the enzyme, and dissociation constants are comparable to the appropriate parameters (Km or Ki) obtained from kinetic measurements. PME-azaG inhibits both the phosphorolytic and synthetic pathway of the reaction in a competitive mode. The complex of 8-azaG with PNP is much weaker than the previously reported Gua-PNP complex, and its dissociation constant increases at pH > 7, where 8-azaG exists predominantly as the monoanion (pKa ≈ 6.5). The fluorescence difference spectrum of the PNP/8-azaG complex points to participation of the N(7)H or/and N(8)H tautomers of the neutral substrate, and the 9-(2-phosphonylmethoxyethyl) derivative also exists as a neutral species in the complex with PNP. The latter conclusion is based on spectral characteristics of the PNP/PME-azaG complex, confirmed by fluorimetric determination of dissociation constants, which are virtually pH-independent in the range 6-7. These findings testify to involvement of the neutral purine molecule, and not its monoanion, as the substrate in the reverse, synthetic reaction. It is proposed that, in the reverse reaction pathway, the natural purine substrate is bound to the enzyme as the neutral N(7)H tautomer, which is responsible for the reported strong fluorescence of the guanine-PNP complex.

Interactions of Calf Spleen Purine Nucleoside Phosphorylase with Antiviral Acyclic Nucleoside Phosphonate Inhibitors: Kinetics and Emission Studies

Association between calf spleen purine nucleoside phosphorylase and a series of phosphonylalkoxyalkyl derivatives of purine bases was studied by inhibition kinetics and fluorimetric titrations. Dissociation constants, determined by fluorimetric titration in phosphate-free conditions, were lower than inhibition constants in 1 mM phosphate, and inhibition was still weaker in 50 mM phosphate, in accord with the postulated bisubstrate analogue character of this class of inhibitors.