Xanthosine and xanthine. Substrate properties with purine nucleoside phosphorylases, and relevance to other enzyme systems.

Substrate properties of xanthine (Xan) and xanthosine (Xao) for purine nucleoside phosphorylases (PNP) of mammalian origin have been reported previously, but only at a single arbitrarily selected pH and with no kinetic constants. Additionally, studies have not taken into account the fact that, at physiological pH, Xao (pKa = 5.7) is a monoanion, while Xan (pKa = 7.7) is an equilibrium mixture of the neutral and monoanionic forms. Furthermore the monoanionic forms, unlike those of guanosine (Guo) and inosine (Ino), and guanine (Gua) and hypoxanthine (Hx), are still 6-oxopurines. The optimum pH for PNP from human erythrocytes and calf spleen with both Xao and Xan is in the range 5-6, whereas those with Guo and Gua, and Ino and Hx, are in the range 7-8. The pH-dependence of substrate properties of Xao and Xan points to both neutral and anionic forms as substrates, with a marked preference for the neutral species. Both neutral and anionic forms of 6-thioxanthine (pKa = 6.5 +/- 0.1), but not of 2-thioxanthine (pKa = 5.9 +/- 0.1), are weaker substrates. Phosphorolysis of Xao to Xan by calf spleen PNP at pH 5.7 levels off at 83% conversion, due to equilibrium with the reverse synthetic pathway (equilibrium constant 0.05), and not by product inhibition. Replacement of Pi by arsenate led to complete arsenolysis of Xao. Kinetic parameters are reported for the phosphorolytic and reverse synthetic pathways at several selected pH values. Phosphorolysis of 200 micro m Xao by the human enzyme at pH 5.7 is inhibited by Guo (IC50 = 10 +/- 2 micro m), Hx (IC50 = 7 +/- 1 micro m) and Gua (IC50 = 4.0 +/- 0.2 micro m). With Gua, inhibition was shown to be competitive, with Ki = 2.0 +/- 0.3 micro m. By contrast, Xao and its products of phosphorolysis (Xan and R1P), were poor inhibitors of phosphorolysis of Guo, and Xan did not inhibit the reverse reaction with Gua. Possible modes of binding of the neutral and anionic forms of Xan and Xao by mammalian PNPs are proposed. Attention is directed to the fact that the structural properties of the neutral and ionic forms of XMP, Xao and Xan are also of key importance in many other enzyme systems, such as IMP dehydrogenase, some nucleic acid polymerases, biosynthesis of caffeine and phosphoribosyltransferases.     

Xanthine, xanthosine and its nucleotides: solution structures of neutral and ionic forms, and relevance to substrate properties in various enzyme systems and metabolic pathways

The 6-oxopurine xanthine (Xan, neutral form 2,6-diketopurine) differs from the corresponding 6-oxopurines guanine (Gua) and hypoxanthine (Hyp) in that, at physiological pH, it consists of a approximately 1:1 equilibrium mixture of the neutral and monoanionic forms, the latter due to ionization of N(3)-H, in striking contrast to dissociation of the N(1)-H in both Gua and Hyp at higher pH. In xanthosine (Xao) and its nucleotides the xanthine ring is predominantly, or exclusively, a similar monoanion at physiological pH. The foregoing has, somewhat surprisingly, been widely overlooked in studies on the properties of these compounds in various enzyme systems and metabolic pathways, including, amongst others, xanthine oxidase, purine phosphoribosyltransferases, IMP dehydrogenases, purine nucleoside phosphorylases, nucleoside hydrolases, the enzymes involved in the biosynthesis of caffeine, the development of xanthine nucleotide-directed G proteins, the pharmacological properties of alkylxanthines. We here review the acid/base properties of xanthine, its nucleosides and nucleotides, their N-alkyl derivatives and other analogues, and their relevance to studies on the foregoing. Included also is a survey of the pH-dependent helical forms of polyxanthylic acid, poly(X), its ability to form helical complexes with a broad range of other synthetic homopolynucleotides, the base pairing properties of xanthine in synthetic oligonucleotides, and in damaged DNA, as well as enzymes involved in circumventing the existence of xanthine in natural DNA.     

Structure and tautomerism of the neutral and monoanionic forms of 2-thiouracil, 2,4-dithiouracil, their nucleosides, and some related derivatives

Ultraviolet and infrared spectrophotometric techniques have been utilized to demonstrate that the monoanionic form of 2-thiouracil in aqueous medium consists of an equilibrium mixture of two tautomeric monoanions, one due to dissociation of the N1 proton, the other to dissociation of the N3 proton, in the approximate ratio 1:1. In contrast to 2,4-diketopyrimidines, and 4-thiouracil, where monoanion formation involves charge delocalization, the two tautomeric monoanions of 2-thiouracil appear to have the charge localized on the O4 position. The neutral forms of 2,4-dithiouracil and 2,4-dithiouridine are in the dithione form in both aqueous and non-aqueous media. The monoanionic form of 2,4-dithiouracil consists of a mixture of two tautomeric monoanions, the predominant one of which is that with the proton on the ring N3, and with charge delocalization on both isomeric monoanions. Such charge delocalization is also present in the monoanion of 2,4-dithiouridine. For the reference compound 2-methylthiopyrimidone-4, the dominant, virtually exclusive, form in chloroform is that with the hydrogen localized on the ring N3, whereas in aqueous medium there is a 1:1 equilibrium mixture of two neutral tautomeric forms, one with the hydrogen on N3, the other with the hydrogen on N1.     

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

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 approximately 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.     

Radical scavenging properties of genistein

The reactivity of genistein toward reactive radical species has been investigated by means of pulse radiolysis. The values of rate constants, respectively 2.3 x 10(10) M(-1)s(-1) and 1.3 x 10(10) M(-1)s(-1) for the reaction with hydroxyl radical at pH 8.3 and 3.0, are close to diffusion limit indicating that genistein is a potent hydroxyl radical scavenger. The reactivity of genistein towards one-electron oxidants has also been investigated. The rate constants k = 4.6 x 10(9) M(-1)s(-1) (pH 8.3) and 6.7 x 10(8) M(-1)s(-1) (pH 7.6) have been determined for the reaction of genistein with *N3 and Br2*- radicals, respectively. For both oxidants the rate constants at pH 3 does not exceed 10(8) M(-1)s(-1). The differences in reactivity of genistein towards the oxidants at different acidity of the solution have been assumed to arise from the acid-base equilibria of genistein. The dissociation constants for genistein (pKa: 7.2, 10.0, and 13.1) have been evaluated spectroscopically. The influence of acid-base equilibria on bond dissociation energy and ionization potential for genistein has also been investigated by means of DFT calculations. It has been concluded on the basis of these calculations that monoanionic form of genistein existing at physiological pH is more powerful radical scavenger than the neutral molecule.     

Prototropic equilibria, tautomerization and electronic absorption properties of dibenzofluorescein in aqueous solution related to its capability as a fluorescence probe

The protolytic equilibria of 1,2,7,8-dibenzofluorescein in aqueous solution have been characterized by visible absorption and fluorescence spectra. The species involved are identified as dianion, monoanion, neutral form and cation. The neutral form includes both the quinoid and lactone structures. The pK(a)s were calculated by an improved procedure to be 3.14, 4.04 and 6.28, respectively. The absorption spectra for each protolytic form were resolved. The absorption maxima (molar absorption coefficient, x10(5), M(-1) cm(-1)) are 532 nm (0.87) for the dianion, 510 nm (0.39) for the monoanion, 500 nm (0.16) for the neutral form, and 494 nm (0.19) for the cation, respectively. Contrary to the assumption in the literature, we found that the monoanion is highly fluorescent (Phi(f) = 0.66, compared to Phi(f) = 0.25 for dianion) and its molar ratio can reach 50% at neutral pH. It is therefore concluded that under physiological pH conditions the monoanion plays a major role when it is used as a fluorescence probe.     

Interaction of 5-fluoro-4-thio-2'-deoxyuridine 5'-phosphate with mammalian tumour thymidylate synthase: role of the pyrimidine N(3)-H dissociation

The role of the pyrimidine N(3)-H in binding of dUMP derivatives to thymidylate synthase was evaluated with the aid of a new dUMP analogue, 5-fluoro-4-thio-dUMP, synthesized by an improved thiation and enzymatic phosphorylation. The interaction of this analogue, and of 5-FdUMP, with the enzyme, and the pH-dependence of these interactions, were compared. Both were slow-binding competitive inhibitors of the enzyme from Ehrlich carcinoma, L1210 and CCRF-CEM cells, with Ki an order of magnitude higher for 5-fluoro-4-thio-dUMP than for 5-FdUMP. With both nucleotides, as well as the parent nucleosides, enzyme inactivation increased as the pH was lowered from 8 to 6. Maximum inactivation with 5-FdUrd was at pH 7.0, and with 5-fluoro-4-thio-dUrd at pH 6.0, in agreement with the higher pKa for the N(3)-H dissociation of the former, and pointing to participation of the N(3)-H as a hydrogen donor in binding to the enzyme.     

New class of 19F pH indicators: fluoroanilines

The pH dependence of the 19F chemical shift has been characterized for a number of fluorine-substituted aniline derivatives. These compounds constitute a new class of 19F nuclear magnetic resonance (NMR) pH indicators, characterized by single 19F resonance lines with sensitivities ranging from 2 to 7 ppm/pH unit near the aniline pKa; total shifts between conjugate acid and base of 5-15 ppm; and pKas ranging from 1 to 7. One compound, N,N-(methyl-2-carboxyisopropyl)-4-fluoroaniline, has a pKa of 6.8 and a sensitivity of 5 ppm/pH unit. This compound displays significant broadening of its 19F resonance near the aniline pKa (6.8), due to a decreased rate of exchange between conjugate acid and base species. Our results are consistent with slow dissociation of an intramolecular hydrogen bond in the zwitterionic species that limits the exchange rate between protonated and unprotonated forms for N,N-(methyl-2-carboxyisopropyl)-4-fluoroaniline.     

Spectroscopic and kinetic evidence for the thiolate anion of glutathione at the active site of glutathione S-transferase

Ultraviolet difference spectroscopy of the binary complex of isozyme 4-4 of rat liver glutathione S-transferase with glutathione (GSH) and the enzyme alone or as the binary complex with the oxygen analogue, gamma-L-glutamyl-L-serylglycine (GOH), at neutral pH reveals an absorption band at 239 nm (epsilon = 5200 M-1 cm-1) that is assigned to the thiolate anion (GS-) of the bound tripeptide. Titration of this difference absorption band over the pH range 5-8 indicates that the thiol of enzyme-bound GSH has a pKa = 6.6, which is about 2.4 pK units less than that in aqueous solution and consistent with the kinetically determined pKa previously reported [Chen et al. (1988) Biochemistry 27, 647]. The observed shift in the pKa between enzyme-bound and free GSH suggests that about 3.3 kcal/mol of the intrinsic binding energy of the peptide is utilized to lower the pKa into the physiological pH range. Apparent dissociation constants for both GSH and GOH are comparable and vary by a factor of less than 2 over the same pH range. Site occupancy data and spectral band intensity reveal large extinction coefficients at 239 nm (epsilon = 5200 M-1 cm-1) and 250 nm (epsilon = 1100 M-1 cm-1) that are consistent with the existence of either a glutathione thiolate (E.GS-) or ion-paired thiolate (EH+.GS-) in the active site. The observation that GS- is likely the predominant tripeptide species bound at the active site suggested that the carboxylate analogue of GSH, gamma-L-glutamyl-(D,L-2-aminomalonyl)glycine, should bind more tightly than GSH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of local anesthetic inhibition of neuronal sodium currents. pH and hydrophobicity dependence.

This study assesses the importance of local anesthetic charge and hydrophobicity in determining the rates of binding to and dissociation from neuronal Na channels. Five amide-linked local anesthetics, paired either by similar pKa or hydrophobicity, were chosen for study: lidocaine, two tertiary amine lidocaine homologs, a neutral lidocaine homolog, and bupivacaine. Voltage-clamped nodes of Ranvier from the sciatic nerve of Bufo marinus were exposed to anesthetic externally, and use-dependent ("phasic") block of Na current was observed. Kinetic analysis of binding (blocking) rates was performed using a three parameter, piecewise-exponential binding model. Changes in extracellular pH (pHo) were used to assess the role of drug protonation in determining the rate of onset of, and recovery from, phasic block. For those drugs with pKa's in the range of pHo tested (6.2-10.4), the forward binding rate during a depolarizing pulse increased at higher pH, consistent with an increase in either intracellular or intramembrane concentration of drug. The rate for unbinding during depolarization was independent of pHo. The dissociation rate between pulses also increased at higher pHo. The pHo dependence of the dissociation rate was not consistent with a model in which the cation is trapped relentlessly within a closed channel. Quantitative estimates of dissociation rates show that the cationic form of lidocaine dissociates at a rate of 0.1 s-1 (at 13 degrees C); for neutral lidocaine, the dissociation rate is 7.0 s-1. Furthermore, the apparent pKa of bound local anesthetic was found to be close to the pKa in aqueous solution, but different than the pKa for "free" local anesthetic accessible to the depolarized channel.     

Probing the mechanism of purine nucleoside phosphorylase by steady-state kinetic studies and ligand binding characterization determined by fluorimetric titrations

Reversible reaction catalyzed by trimeric purine nucleoside phosphorylase (PNP) from Cellulomonas sp. with typical and non-typical substrates, including product inhibition patterns of both reaction directions, and interactions of the enzyme with bisubstrate analogue inhibitors, were investigated by the steady-state kinetic methods and fluorimetric titrations. The ligand chromophores exist most probably as neutral species, and not N(1)-H monoanions, in the complex with PNP, as shown by determination of inhibition constants vs. pH. This supports the mechanism in which hydrogen bond interaction of N(1)-H with Glu204 is crucial in the catalytic process. Stoichiometry of ligand binding, with possible exception of hypoxanthine, is three molecules per enzyme trimer. Kinetic experiments show that in principle the Michaelis-Menten model could not properly describe the reaction. However, this model seems to hold for certain experimental conditions. Data presented here are supported by earlier findings obtained by means of fluorimetric titrations and protective effects of ligands on thermal inactivation of the enzyme. All results are consistent with the following mechanism for trimeric PNPs: (i) random binding of substrates, (ii) potent binding and slow release of some reaction products leading to the circumstances that the chemical step is not the slowest one and that rapid-equilibrium assumptions do not hold, (iii) a dual role of phosphate--a substrate and also a reaction modifier.     

Proton transfer kinetics in the lowest excited state of 1,N6-ethenoadenosine as revealed by fluorescence lifetime measurements

Fluorescence lifetimes have been examined of 1, N6-ethenoadenosine (epsilon Ado), 1-methyl-ethenoadenosine (m1 epsilon Ado+), 9-methyl-ethenoadenosine (m9 epsilon Ado+), N1-deazaethenoadenosine (N1-deaza-epsilon Ado, and 9-methyl-N1-deazaethenoadenosine (m9N1-deaza epsilon Ado) at various pH's in the 7.5 approximately 1.5 region and at 20 degrees C. From an analysis we have reached the following conclusions. (1) The observed fluorescence of epsilon Ado is caused by the unprotonated, neutral, excited species epsilon Ado not not only at pH 7 but also at pH 2. (2) pKa of the excited epsilon Ado is lower than that of the ground state epsilon Ado. (3) The rate constant of protonation in the excited state is about 10(10) sec-1 M-1.     

Recognition modes of hypoxanthine,xanthine and their derivatives by amino acid carboxylic group: UV spectroscopic and quantum chemical data

UV absorption spectra of Hyp, Xan, their nucleosides and methyl derivatives were studied in anhydrous dimethylsuloxide and the changes in these spectra on the interactions with neutral and deprotonated carboxylic groups of amino acids were traced. By the semiempirical quantum-chemical method MNDO/H it was shown, that interaction with carboxylate-ion fixes Hyp as the rare N7H enolic tautomer and converts Xan into its N9H diketo tautomeric form with a probable admixture of the N7H O6-enolic form. Significant changes in the UV spectra of Xan, m3Xan, m9Xan and X under interaction with carboxylate-ion are determined by essential contribution to complex formation of proton transfer from bases to ligands, m9Xan and X proving to be slightly protonated even by the solvent. The methylation of the N7 position in m7I and m7X was established to result in the practical absence of their interactions with carboxylate-ion and initiation of a new ability of forming complex with the neutral carboxylic group. Substitution of the C8H group by N in 8-azaXan does not change the interaction specificity of the base with two forms of carboxylic group.     

Probing electrostatic interactions along the reaction pathway of a glycoside hydrolase: histidine characterization by NMR spectroscopy

We have characterized by NMR spectroscopy the three active site (His80, His85, and His205) and two non-active site (His107 and His114) histidines in the 34 kDa catalytic domain of Cellulomonas fimi xylanase Cex in its apo, noncovalently aza-sugar-inhibited, and trapped glycosyl-enzyme intermediate states. Due to protection from hydrogen exchange, the level of which increased upon inhibition, the labile 1Hdelta1 and 1H epsilon1 atoms of four histidines (t1/2 approximately 0.1-300 s at 30 degrees C and pH approximately 7), as well as the nitrogen-bonded protons in the xylobio-imidazole and -isofagomine inhibitors, could be observed with chemical shifts between 10.2 and 17.6 ppm. The histidine pKa values and neutral tautomeric forms were determined from their pH-dependent 13C epsilon1-1H epsilon1 chemical shifts, combined with multiple-bond 1H delta2/epsilon1-15N delta1/epsilon2 scalar coupling patterns. Remarkably, these pKa values span more than 8 log units such that at the pH optimum of approximately 6 for Cex activity, His107 and His205 are positively charged (pKa > 10.4), His85 is neutral (pKa < 2.8), and both His80 (pKa = 7.9) and His114 (pKa = 8.1) are titrating between charged and neutral states. Furthermore, upon formation of the glycosyl-enzyme intermediate, the pKa value of His80 drops from 7.9 to <2.8, becoming neutral and accepting a hydrogen bond from an exocyclic oxygen of the bound sugar moiety. Changes in the pH-dependent activity of Cex due to mutation of His80 to an alanine confirm the importance of this interaction. The diverse ionization behaviors of the histidine residues are discussed in terms of their structural and functional roles in this model glycoside hydrolase.     

Binding of (6R,S)-methyltetrahydrofolate to methyltransferase from Clostridium thermoaceticum: role of protonation of methyltetrahydrofolate in the mechanism of methyl transfer

The methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoacetium catalyzes transfer of the N5-methyl group of (6S)-methyltetrahydrofolate (CH3-H4folate) to the cob(I)amide center of a corrinoid/iron-sulfur protein (CFeSP), forming H4folate and methylcob(III)amide. We have investigated binding of 13C-enriched (6R,S)-CH3-H4folate and (6R)-CH3-H4folate to MeTr by 13C NMR, equilibrium dialysis, fluorescence quenching, and proton uptake experiments. The results described here and in the accompanying paper [Seravalli, J., Shoemaker, R. K., Sudbeck, M. J., and Ragsdale, S. W. (1999) Biochemistry 38, 5728-5735] constitute the first evidence for protonation of the pterin ring of CH3-H4folate. The pH dependence of the chemical shift in the 13C NMR spectrum for the N5-methyl resonance indicates that MeTr decreases the acidity of the N5 tertiary amine of CH3-H4folate by 1 pK unit in both water and deuterium oxide. Binding of (6R,S)-CH3H4folate is accompanied by the uptake of one proton. These results are consistent with a mechanism of activation of CH3-H4folate by protonation to make the methyl group more electrophilic and the product H4folate a better leaving group toward nucleophilic attack by cob(I)amide. When MeTr is present in excess over (6R,S)-13CH3-H4folate, the 13C NMR signal is split into two broad signals that reflect the bound states of the two diastereomers. This unexpected ability of MeTr to bind both isomers was confirmed by the observation of MeTr-bound (6R)-13CH3-H4folate by NMR and by the measurement of similar dissociation constants for (6R)- and (6S)-CH3-H4folate diastereomers by fluorescence quenching experiments. The transversal relaxation time (T2) of 13CH3-H4folate bound to MeTr is pH independent between pH 5.50 and 7.0, indicating that neither changes in the protonation state of bound CH3-H4folate nor the previously observed pH-dependent MeTr conformational change contribute to broadening of the 13C resonance signal. The dissociation constant for (6R,S)-CH3-H4folate is also pH independent, indicating that the role of the pH-dependent conformational change is to stabilize the transition state for methyl transfer, and not to favor the binding of CH3-H4folate.     

Formation of 2-chloroinosine from guanosine by treatment of HNO(2) in the presence of NaCl

We investigated the reaction of Guo with nitrous acid in the presence of NaCl. When 1 mM Guo was incubated with 100 mM NaNO(2) and 2M NaCl in sodium acetate buffer at pH 3.2 and 37 degrees C, 2-chloroinosine (2-Cl-Ino) was produced in addition to oxanosine (Oxo) and xanthosine (Xao). The yield of 2-Cl-Ino was 0.033 mM at an incubation time of 2 h. Under the same reaction conditions, GMP and dGuo gave rise to the corresponding 2-chloro derivatives with comparable yields. All the 2-chloro derivatives were fairly stable (t(12)>360 h) at physiological pH and temperature. To elucidate the reaction mechanism of the chlorination, the diazoate derivative of Guo, a reaction intermediate of the Guo-HNO(2) system, was employed as a starting compound. When the diazoate was incubated with 2M NaCl in a neutral solution, 2-Cl-Ino was produced in addition to Oxo and Xao. These results suggest that the 2-chloro derivatives can be produced from foodstuffs in the human stomach and may have potential importance as a carcinogen causing gastric cancer.     

Substrate specificity and protonation state of Escherichia coli ornithine transcarbamoylase as determined by pH studies. Binding of carbamoyl phosphate

Binding of carbamoyl phosphate to Escherichia coli ornithine transcarbamoylase and its relation to turnover have been examined as a function of pH under steady-state conditions. The pH profile of the dissociation constant of carbamoyl phosphate (Kiacp) shows that the affinity of the substrate increases as pH decreases. Two ionizing groups are involved in carbamoyl phosphate binding. Protonation of an enzymic group with pKa 9.6 results in productive binding of the substrate with a moderate affinity of Kiacp approximately 30 microM. Protonation of a second group further enhances binding by roughly another order of magnitude. This ionization occurs with a pKa that shifts from less than 6 in the free enzyme to 7.3 in the binary complex. However, tighter binding of carbamoyl phosphate due to this ionization does not contribute to catalysis. The turnover rate (kcat) of the enzyme diminishes in the acidic pH range and is governed by an ionization with a pKa of 7.2. Both the catalytic pKa of 7.2 and the productive binding pKa of 9.6 appear in the pH profile of kcat/KMcp. Together with earlier kinetic results (Kuo, L. C., Herzberg, W., and Lipscomb, W. N. (1985) Biochemistry 24, 4754-4761), these data suggest that the step which modulates kcat may occur prior to the binding of the second substrate L-ornithine.     

Dissociation constants for dihydrofolic acid and dihydrobiopterin and implications for mechanistic models for dihydrofolate reductase

The dissociation constants (pKa) for the pteridine ring system of dihydrofolate (H2folate) have been redetermined, and those for dihydrobiopterin (H2biopterin) have been determined. Determination of the pKa for N5 of H2folate is complicated by the low solubility and instability of H2folate at pH 2-4, and other complicating factors. The initial rate of absorbance change due to degradation is a maximum at pH 2.5, and the products depend on the oxygen concentration: under aerobic conditions, (p-aminobenzoyl)glutamic acid and 7,8-dihydropterin-6-carboxaldehyde are major products. H2Biopterin is much more soluble and more stable at low pH. For protonation of N5, the pKa is 2.56 +/- 0.01 for H2biopterin and 2.59 +/- 0.03 for H2folic acid. Spectrophotometric determination of the pKa for the N3-O4 amide group of H2folate is subject to serious errors when a wavelength between 220 and 235 nm is used. These errors arise from the pH-dependent absorbance of mercaptoethanol often present in the preparation. The amide group has a pKa of 10.41 +/- 0.04 in H2biopterin and 10.85 +/- 0.04 in H2folate. The redetermined value for the pKa of N5 of H2folate has implications for mechanistic models for dihydrofolate reductase, and revised kinetic constants have been calculated for one model.     

Substrate recognition and activation mechanism of D-amino acid oxidase: a study using substrate analogs

We investigated the mechanism of recognition and activation of substrate by D-amino acid oxidase (DAO) by thermodynamical and spectrophotometric methods using zwitterionic ligands [N-methylisonicotinate (NMIN), trigonelline, and homarine] and monoanionic ligands as model compounds of the substrate and the product. In terms of the charge within the substrate D-amino acid, monoanionic (e.g., benzoate), zwitterionic (e.g., NMIN), and dianionic (e.g., terephthalate) ligands are thought to be good models for neutral, basic, and acidic amino acids, respectively, because when a substrate binds to DAO, as previously reported, the a-ammonium group (-NH(3)(+)) probably loses a proton to become neutral (-NH(2)) before the oxidation. Zwitterionic ligands can also be good model compounds of product in the purple complex (the complex of reduced DAO with the product imino acid), because the imino nitrogen of the imino acid is in a protonated cationic form. We also discuss electrostatic interaction, steric effect, and charge-transfer interaction as factors which affect the affinity of substrate/ligand for DAO. Monoanionic ligands have high affinity for neutral forms of oxidized and semiquinoid DAO, while zwitterionic ligands have high affinity for anionic forms of oxidized, semiquinoid, and reduced DAO; this difference was explained by the electrostatic interaction in the active site. The low affinity of homarine (N-methylpicolinate) for oxidized DAO, as in the case of o-methylbenzoate, is due to steric hindrance: one of the ortho carbons of benzoate is near the phenol carbons of Tyr228 and the other ortho carbon is near the carbonyl oxygen of Gly313. The correlation of the affinity of meta- and para-substituted benzoates for oxidized DAO with their Hammet's s values are explained by the HOMO-LUMO interaction between the phenol group of Tyr224 and the benzene ring of benzoate derivative. The pK(a) of neutral flavin [N(3)-H of oxidized flavin, N(5)-H of semiquinoid flavin, and N(1)-H of reduced flavin] decreases by its binding to the apoenzyme. The magnitude of the decrement is oxidized flavin < semiquinoid flavin < reduced flavin. The largest factor in the substantially low pK(a) of reduced flavin in DAO is probably the steric hindrance between the hydrogen atom of H-N(1)(flavin) and the hydrogen atom of H-N of Gly315, which becomes significant when a hydrogen is bound to N(1) of flavin.     

Inhibition of the elastase of Pseudomonas aeruginosa by N alpha-phosphoryl dipeptides and kinetics of spontaneous hydrolysis of the inhibitors

The rates of hydrolysis of N-[(alpha-L-rhamnopyranosyloxy)phospho]-L-leucyl-L-tryptophan (phosphoramidon), N alpha-phosphoryl-L-leucyl-L-tryptophan (PO3LeuTrp), N alpha-phosphoryl-L-leucyl-L-phenylalanine (PO3LeuPhe), and N alpha-phosphoryl-L-leucyl-L-phenylalaninamide (PO3LeuPheNH2) were followed by proton nuclear magnetic resonance spectroscopy. The rates of hydrolysis (kobsd) of PO3LeuTrp, PO3LeuPhe, and PO3LeuPheNH2 were all first order in phosphorylamide concentration over the pH range studied (3.8-9.5). The values for kobsd at pH 7.3 and 37 degrees C are as follows: PO3LeuTrp, 0.35 h-1; PO3LeuPhe, 0.63 h-1; PO3LeuPheNH2, 0.73 h-1. The values for kobsd do not significantly change between pH 5 and pH 8 but dramatically decreased with increasing pH. The hydrolysis of PO3LeuPhe and PO3LeuPheNH2 above a pH of approximately 5 was positively correlated with the concentration of monoanionic species (NHRPO3H)1-, and the values for the first-order rate constants for the respective monoanionic species were calculated to be 0.66 +/- 0.03 h-1 and 1.07 +/- 0.10 h-1. Phosphoramidon was not found to hydrolyze after 6 days at 37 degrees C at a pH of 4.6 and 7.7, while the phosphorylamide PO3LeuTrp, synthesized by the removal of L-rhamnose from phosphoramidon by base hydrolysis, was found to rapidly hydrolyze under these conditions. Solvolysis in aqueous methanol of PO3LeuPhe and PO3LeuPheNH2 indicates that the hydrolysis reaction is bimolecular, proceeding by way of direct attack of solvent (H2O, CH3OH) on phosphorus.(ABSTRACT TRUNCATED AT 250 WORDS)