Hydrolysis of p-nitrotrifluoroacetanilide catalyzed by water and imidazole

The hydrolyses of p-nitrotrifluoroacetanilide catalyzed by water and imidazole were examined at 70°C. The pH-rate constant profile of the hydrolysis in H₂O was examined in the pH range 0.0–11.4. The hydrolysis was independent of pH in the region from pH 1.0 to 4.5, presumably a water-catalyzed reaction. The rate constant and the D₂O solvent isotope effect for this reaction were 1.0 × 10^?4 sec^?1 and 3.7, respectively. Both natural imidazole and imidazolium cation catalyzed hydrolysis. The rate constant of the hydrolysis catalyzed by neutral imidazole was determined to be 5.4 × 10^?3M^?1 sec^?1 and the D₂O solvent isotope effect was 1.8.     

Synthesis and stereochemistry of new cobalt azo-nitroso complexes

Synthesis and characterization of new cobalt-substituted phenylazo 2,4-dinitrosoresorcinol complexes have been carried out. The analytical data depict the formation of complex compounds with the stoichiometry 2:3 (o-COOH, m-NO₂) and 1:2 (o-Cl, o-CH₃, m-Cl, m-CH₃). All the complexes are of low spin in octahedral and square planar or distorted tetrahedral environments. The octahedral ⇄ tetrahedral equilibria are evident. The complexes in the presence of basic compounds gave some addition products. The electronic transitions and the ligand field parameters are assigned and calculated. The complex formation occurred through the azo group and the phenolic oxygen atom in most complexes. In the o-carboxy ligand, the COO⁻, NN and the oximic groups participate through complexation.     

Chemical induction of poly-cis carotenoid biosynthesis

A new class of synthetic bioregulators is reported which cause the accumulation of poly-cis carotenoids in the flavedo of Marsh white seedless grapefruit. The compounds tested were all secondary amines: dibenzylamine, substituted dibenzylamines (4-F; 4-Cl; 4-Br; 2-, 3-and 4-Me; 4-NO₂; 4-CN; 4-Cl, 4′-Me; 4-Me, 4′-NO₂), N-benzyl phenethylamine and N-benzyl 2-naphthalenemethylamine. The most effective, 4-chlorodibenzylamine, caused the accumulation of 74μgg/g dry wt of poly-cis carotenoids. Prolycopene was the predominant pigment but substantial amounts of proneurosporene, poly-cis-γ-carotenes and other cis carotenes were also present. The mode of action of these new bioregulators is probably gene derepression, the same as that of the lycopene inducers. However, the secondary amines probably derepress a recessive gene governing the biosynthesis of poly-cis carotenoids; whereas, the lycopene inducers derepress the dominant gene that gives rise to the normal all-trans carotenoids. The new compounds did not seem to inhibit the cyclase(s), as the lycopene inducers do.     

Synthesis and antitubercular activity of novel Schiff bases derived from d-mannitol

Six Schiff base derivatives of d-mannitol, 1,6-dideoxy-1,6-bis-{[(E)-arylmethylidene]amino}-d-mannitol (6: aryl = XC₆H₄: X = o-, m- and p- Cl or NO₂), have been synthesized and evaluated for their in vitro antibacterial activity against Mycobacterium tuberculosis H₃₇Rv using the Alamar Blue susceptibility test and the activity expressed as the minimum inhibitory concentration (MIC) in μg/mL. All three nitro derivatives exhibit significant activities: activities of (6d: X = o-NO₂), (6e: X = m-NO₂) and (6f: X = p-NO₂) are 12.5, 25.0 and 25.0 μg/mL, respectively. When compared with first line drugs, such as ethambutol, they can be considered as a good starting point to develop new lead compounds for the treatment of multidrug-resistant tuberculosis. Characterization of the new compounds 6 is generally achieved spectroscopically. The structure of compound 3 has been confirmed by X-ray crystallography.     

Effect of Castanospermine and Related Polyhydroxyalkaloids on Purified Myrosinase from Lepidium sativum Seedlings

Myrosinase (β-thioglucoside glucohydrolase, EC 3.2.3.1) was purified to apparent homogeneity from light-grown cress (Lepidium sativum L.) seedlings. This enzyme, which catalyzes hydrolysis of the glucosinolate sinigrin (K_m, 115 micromolar) at an optimum pH of 5.5 in sodium citrate buffer, had a native molecular weight of 130 ± 5 kilodaltons and an isoelectric point of 4.7 to 4.9. SDS-PAGE revealed two polypeptides with molecular weights of 62 and 65 kilodaltons. Both subunits contained carbohydrate as shown by periodic acid-Schiff staining. The purified enzyme hydrolyzed p-nitrophenyl-β-d-glucoside (K_m, 2.0 millimolar) at an optimum pH of 6.5 in phosphate buffer. The indolizidine alkaloid castanospermine, a known inhibitor of O-glycosidases, competitively inhibited the hydrolyses of sinigrin (thioglucosidase activity) and p-nitrophenyl-β-d-glucoside (O-glucosidase activity) with K_i values of 5 and 6 micromolar, respectively. In contrast, the related polyhydroxyalkaloids swainsonine and deoxynojirimycin were without effect upon these hydrolyses.     

Mechanism of action of chalcone isomerase

The pH profile of the rate of isomerization of 4,2′,4′-trihydroxychalcone catalyzed by chalcone isomerase shows dependence on the basic form of a group with a pK of 7.25. The same pH dependence is seen for the reverse reaction. Enzyme activity is lost in the presence of diethylpyrocarbonate at pH 6.0. In the presence of 20% formamide in imidazole buffers, the pK for the forward reaction is modified by a second pK of 7.1. This behavior represents a perturbed pK of a neutral acid group and is attributable to the 2′ hydroxyl of the chalcone substrate. These results suggest a mechanism of enzyme action involving nucleophilic addition of an imidazole group in the active site to the double bond followed by nucleophilic attack by the 2′ phenolate group, resulting in ring closure with inversion of configuration at C-2.     

The kinetics of papain- and ficin-catalysed hydrolyses in the presence of alcohols

1. The maximum rate of production of p-nitrophenol (V_max.) for both papain- and ficin-catalysed hydrolyses of p-nitrophenyl hippurate is independent of methanol concentration up to 2m for papain and 1·5m for ficin. 2. The observed catalytic constant (k₀) for the production of hippuric acid for both papain- and ficin-catalysed hydrolyses of methyl hippurate decreases with increasing methanol concentration, 1/k₀ being linearly dependent on the methanol concentration. The k_MeOH/k_H2O ratio is determined. 3. These results provide strong evidence against general base catalysis for the rate-determining step in the deacylation of hippuryl-papain and hippuryl-ficin and probably for other specific acyl-papains and acyl-ficins. 4. The rate-determining step for the deacylation of the non-specific trans-cinnamoyl-papain appears to be different from that for the specific hippuryl-papain, and is probably subject to general base catalysis. It is possible, however, to accommodate all these observations in a single four-step reaction pathway. 5. Propan-2-ol did not influence the rate of production of hippuric acid for the papain-catalysed hydrolysis of methyl hippurate. A similar result has previously been reported for the ficin-catalysed hydrolysis of methyl hippurate. Ethanol and of course methanol (see 2) decrease the rate of production of hippuric acid for both papain- and ficin-catalysed hydrolyses of methyl hippurate. It is suggested that the secondary alcohol is incapable for structural reasons of approaching the bond to be hydrolysed.     

Disubstituted diaryl diselenides inhibit δ-ALA-D and Na+, K+-ATPase activities in rat brain homogenates in vitro

Toxicological and pharmacological studies demonstrated that the introduction of functional groups into the aromatic ring of diphenyl diselenide alter its effect. The aim of this study was to evaluate the in vitro effect of m-trifluoromethyl-diphenyl diselenide (m-CF₃–C₆H₄Se)₂, p-chloro-diphenyl diselenide (p-Cl–C₆H₄Se)₂ and p-methoxyl-diphenyl diselenide (p-CH₃O–C₆H₄Se)₂ on δ-aminolevulinate dehydratase (δ-ALA-D) and Na⁺, K⁺-ATPase activities in rat brain homogenates. Diselenides inhibited δ-ALA-D activity (IC₅₀ 4–6 μM [concentration inhibiting 50%]), and dithiothreitol (DTT) restored the enzyme activity. ZnCl₂ (100 μM) did not restore δ-ALA-D inhibition caused by (p-Cl–C₆H₄Se)₂ and (m-CF₃–C₆H₄Se)₂. Na⁺, K⁺-ATPase activity was more sensitive to (p-Cl–C₆H₄Se)₂ and (m-CF₃–C₆H₄Se)₂ (IC₅₀ 6 μM) than (p-CH₃O–C₆H₄Se)₂ and (PhSe)₂ (IC₅₀ 45 and 31 μM, respectively). DTT restored the activity of Na⁺, K⁺-ATPase inhibited by diselenides. The effect of diselenides on Na⁺/K⁺-ATPase is dependent on their substitutions in the aromatic ring. The mechanism through which diselenides inhibit δ-ALA-D and Na⁺, K⁺-ATPase activities involves the oxidation of thiol groups.     

Kinetics of reaction of square-planar complexes of nitrosubstituted aromatic di-imines with methoxide and aliphatic amines in dipolar aprotic solvents

The square-planar complexes [Pt(5-NO₂phen)Cl₂], [Pd(5-NO₂phen)₂](ClO₄)₂ and [Pd(5-NO₂phen)X₂] (X = N₃, Cl) give a colour forming reaction with OH⁻ or alkoxides in dipolar aprotic solvents. The process occurs in one observable step and the rate law is k_obs = k₂ [alkoxide]. The second order rate constant k₂ is independent of the nature of the metal and of the coordinated ligands other than 5-NO₂-phen and this is taken as evidence that the nucleophilic attack does not occur at the central atom but rather at the coordinated phenanthroline to give presumably σ-adducts at the carbon. Conductivity stopped-flow shows that the colour forming reaction between neutral substrates and amines is associated with the formation of a 1:1 electrolyte. In dipolar aprotic solvents a colour forming reaction occurs also between [Pd(4-NO₂bipy)Cl₂] and bases.     

Synthesis and evaluation of a class of 1,4,7-triazacyclononane derivatives as iron depletion antitumor agents

Iron depletion has been confirmed as an efficient strategy for cancer treatment. In the current study, a series of 1,4,7-triazacyclononane derivatives HE-NO2A, HP-NO2A and NE2P2A, as well as the bifunctional chelators p-NO₂-PhPr-NE3TA and p-NH₂-PhPr-NE3TA were synthesized and evaluated as iron-depleting agents for the potential anti-cancer therapy against human hepatocellular carcinoma. The cytotoxicity of these chelators was measured using hepatocellular cancer cells and compared with the clinically available iron depletion agent DFO and the universal metal chelator DTPA. All these 1,4,7-triazacyclononane-based chelators exhibited much stronger antiproliferative activity than DFO and DTPA. Among them, chelators with phenylpropyl side chains, represented by p-NO₂-PhPr-NE3TA and p-NH₂-PhPr-NE3TA, displayed the highest antiproliferative activity against HepG2 cells. Hence, these compounds are attractive candidates for the advanced study as iron depletion agents for the potential anti-cancer therapy, and could be further in conjugation with a targeting moiety for the future development in targeted iron depletion therapy.     

Substitution effect on new Co(II) addition compounds with salicylaldehydes and the nitrogenous bases phen or neoc: Crystal and molecular structures of [Co(5-NO2-salicylaldehyde)2(phen)], [Co(5-CH3-salicylaldehyde)2(neoc)] and [Co(5-Cl-salicylaldehyde)2(neoc)]

The cobalt(II) addition compounds [Co(X-salo)₂(Y)], where X-salo is the anion of substituted salicylaldehydes (X = 3-OCH₃, 5-CH₃, 5-Cl, 5-NO₂ and Y = the neutral 1,10 phenanthroline or neocuproine), were synthesized and characterized by physicochemical and spectral (IR, UV-Vis) data. Theoretical calculations (DFT, ZINDO, TD DFT) with gaussian 03 for the prediction of the electronic spectrum for the compounds, gave good correlation with the experimental one in the solid state and in solution. The cyclic voltammetry study in CH₃CN gave all the expected waves for the redox processes of the metal Co(II) and the ligands phen or neoc and salicylaldehydes. The X-ray diffraction study of three compounds [Co(5-NO₂-salo)₂(phen)], [Co(5-CH₃-salo)₂(neoc)] and [Co(5-Cl-salo)₂(neoc)] verified their analogous proposed octahedral arrangement of the ligands around the cobalt(II) atom.     

Hydrolysis of neutral phosphate and phosphonate esters catalysed by Co2+-chelates of tris-imidazolyl phosphines

The hydrolysis of two neutral phosphate esters, (tris-2-pyridylphosphate and tris-p-nitrophenylphosphate, 5 and 4 respectively) and a phosphonate ester (ethyl-p-nitrophenylmethylphosphonate (6)) were studied in the presence of Co²⁺-complexes of two tris- imidazolylphosphines. In the hydrolyses of both 5 and 6, the Co²⁺-complex of bis-[4,5-diisopropyl- imidazol-2-yl]imidazole-2-yl phosphine (2) appears to act as the anionic form, generated by ionization of a Co²⁺-bound H₂O having a pK_a of ～8 at 37 °C in 80% ethanol-H₂O. On the other hand, the Co²⁺-complex of bis-[4,5-diisopropylimidazol-2-yl]-4(5)- hydroxyethylimidazol-2-yl phosphine (3) catalyses the hydrolyses of 5 and 6 with an activity which increases linearly with pH. In this case, the active form of Co²⁺:3 has a pK_a in excess of 8.3 which apparently obtains from ionization of the Co²⁺- associated hydroxyethyl group. Evidence is presented that Co²⁺:3 functions as a general base catalyst in the hydrolysis of 5.     

Dependence on pH of the activity of pig liver esterase

The dependence on pH of the kinetic parameters for the hydrolysis of phenyl acetate catalyzed by pig liver carboxylesterase was examined for purified high-isoelectric point and low-isoelectric point fractions of enzyme that were separated by isoelectric focusing. The values of k_cat are half-maximal at pH 4.3 and 5.1 for the high- and low-isoelectric point forms, respectively, and show a shallow dependence on pH with a value of n = 0.5. The absence of a change in the pH dependence of k_cat for the high-isoelectric point enzyme in the presence of high concentrations of methanol, which reacts with the acetyl-enzyme intermediate to give methyl acetate, provides evidence that the pH dependence is not caused by a change in rate-determining step. This means that if an imidazole group is involved in catalysis its pK must be perturbed downward by 2–3 units. The pH dependence of $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ is biphasic with apparent pK values for dissociations of the free enzyme near 7 and 4 for both the high- and low-isoelectric point enzymes. Inhibition by a second molecule of substrate and by methanol are strongest for high-pH forms of the enzyme.     

Interaction of copper(II) complexes with bis(p-nitrophenyl)phosphate: Structural and spectral studies

When the complexes [Cu(L1)(H₂O)](ClO₄)₂1, where L1 = 4-methyl-1-(pyrid-2-ylmethyl)-1,4-diazacycloheptane, and [Cu(L2)Cl₂] 2, where L2 = 4-methyl-1-(quinol-2-ylmethyl)-1,4-diazacycloheptane are interacted with one/two equivalents of bis(p-nitrophenylphosphate, (p-NO₂Ph)₂PO₂, BNP), no hydrolysis of BNP is observed. From the solution the adducts of copper(II) complexes [Cu₂(L1)₂((p-NO₂Ph)₂PO₂)₂]-(ClO₄)₂3 and [Cu(L2)((p-NO₂Ph)₂PO₂)₂]·H₂O 4 have been isolated and structurally characterised. The X-ray crystal structure of 3 contains two Cu(L1) units bridged by two BNP molecules. The Cu···Cu distance (5.1 Å) reveals no Cu-Cu interaction. On the other hand, the complex 4 is mononuclear with Cu(II) coordinated to the 3N ligand as well as BNP molecules through phosphate oxygen. The trigonality index (τ, 0.37) observed for 4 is high suggesting the presence of significant trigonal distortion in the coordination geometry around copper(II). The complexes are further characterized by spectral and electrochemical studies.     

The reaction of amino alcohols with active esters: II. Catalysis of the acylation step by metal ions

The reaction of triethanolamine (TEA) with active substrates—p-nitrophenyl esters and cinnamoyl imidazole (CI)—is catalyzed by divalent heavy metal ions. With Hg²⁺, rate enhancements of 100–1000 (depending on the substrate) were observed, the overall rate constants of substrate decomposition thus exceeding those of spontaneous hydrolysis up to 100,000-fold. The predominant active species at low L:M ratio was found to be the Hg-(TEA)₂ complex. The dependence of the reaction rate upon excess of amino alcohol—at constant Hg²⁺ concentration—is attributable to formation of another active complex—Hg-(TEA)₃.The high reactivity of the system is due to the alcoholate group of metal-bound TEA, whose pK has been lowered by the proximity of the metal ion. This labile nucleophilic alcoholate attacks the substrate causing its alcoholysis and forming O-acyl-TEA. The lability of the metal-alcoholate bond can be enhanced by low concentrations of halide ions, thus causing up to 5-fold additional increase in alcoholysis rate. Higher halide ion concentrations cause inhibition, probably due to formation of inactive HgX₂ molecules.Presumably an important role of the metal ion in metalloenzymes is to affect the decrease in the pK value of a reactive group so that it can exhibit activity under physiological conditions.     

Partial Purification and Characterization of a 3'- Phosphoadenosine 5' -Phosphosulfate: Desulfoglucosinolate Sulfotransferase from Cress (Lepidium sativum)

A 3′ -phosphoadenosine 5′ -phosphosulfate (PAPS):desulfoglucosinolate sulfotransferase (EC 2.8.2-) was extensively purified from light-grown cress (Lepidium sativum L.) seedlings by gel filtration and concanavalin A-Sepharose 4B, Matrex Gel Green A, and Mono Q fast protein liquid chromatography. The purified enzyme, which required bovine serum albumin for stabilization, had a native molecular weight of 31,000 ± 5,000 and an apparent isoelectric point of 5.2. Using PAPS (K_m 60 micromolar) as sulfur donor, it catalyzed the sulfation of desulfobenzylglucosinolate (K_m 82 micromolar), desulfo-p-hydroxybenzylglucosinolate (K_m 670 micromolar), and desulfoallylglucosinolate (K_m 6.5 millimolar) at an optimal pH of 9.0. All other potential substrates tested, including flavonoids, flavonoid glycosides, cinnamic acids, and phenylacetaldoxime, were not sulfated. Sulfotransferase activity was stimulated by MgCl₂, MnCl₂ and reducing agents and inhibited by ZnCl₂, PbNO₃ NiCl₂ and the reaction product PAP. The thiol reagents N-ethylmaleimide, p-chloromercuriphenylsulfonic acid, and 5,5′ -dithio-bis-(2-nitrobenzoic acid) were also potent inhibitors, but the enzyme was protected from covalent modification by β-mercaptoethanol. The kinetics of desulfobenzylglucosinolate sulfation were consistent with a rapid equilibrium ordered mechanism with desulfobenzylglucosinolate binding first and PAPS second.     

Enantiomer-selective solvolysis catalysed by a histidine-containing cyclic dipeptide carrying chiral side chain

Tripeptides with cyclic dipeptide backbones, cyclo[l-Glu(l-Leu-O Bzl)-t-His] and cyclo[l-Glu(l-Leu-OH)-Ir His, and the corresponding tripeptides with linear backbones, Me₃COCO-l-Glu(l-Leu-OBzl)-l-His-OMe and Me₃COCO-l-Glu(l-Leu-OH)-l-His-OMe, were synthesized and used as catalysts for the hydrolysis of carboxylic acid active esters of various types. The experimental results are summarized as follows. (I) In the hydrolysis of a neutral and hydrophobic substate, p-nitrophenyl laurate, in 20% dioxane/H₂O mixture of pH 7.8, a hydrophobic and flexible peptide, Me₃COCO-l-Glu(l-Leu-OH)-l-His-OMe, was more reactive than imidazole. On the other hand, cyclo[l-Glu(l-Leu-OBzl)-l-His] and cyclo[l-Leu-OH)-l-His], which have rigid backbone chain and fixed sidechain conformation, were not particularly reactive. (2) in the solcolysis of a positively charged substrate, p-nitrophenyl glycinate hydrochloride, in 42% i-PrOH/H₂O mixture at pH 6.95, a positively charged substrate, p-nitrophenyl glycinate hydrochloride, in 42% i-PrOH/H₂O mixture at pH 6.95, a negatively charged and flexible peptide, Me₃COCO-l-Glu(l-Leu-OH)-l-His-OMe, was more reactive than imidazole. However, cyclo [l-Glu(l-Leu-OH)-l-His] was not particularly reactive in the same reaction. In the hydrolysis of p-nitrophenyl glycinate hydrochloride in aqueous solution at pH 7.8 a hydrophobic and rigid peptide, cyclo[(l-Glu(l-Leu-OBzl)-l-His], was more reactive than imidazole. However, in the hydrolysis of p-nitrophenyl CO-AMINODODECANOATE hydrochloride, which has a positive charge and a rective site separated by a long hydrophobic chain, peptide catalysts did not show efficient catalysis. (3) In the hydrolysis of a positively charged, hydrophobic and chiral substrate, p-nitrophenyl leucinate hydrochloride, in aqueous solution at pH 6.95, the d-enantiomer was hydrolysed more quickly that the t-enantiomer with cyclo[l-Glu(l-Leu-OBzl)l-His] or cyclo[t-Glu(l-Leu-OH)-l-His] as catalyst. On the other hand, the tripeptides with linear backbone did not effect an enantiomer-selective catalysis. The solvolytic reaction catalysed by the tripeptides with cyclic dipeptide backbone in 42% i-PrOH/water mixture was also enantiomer-selective.     

The agonist action of substituted phenylethanolamines on octopamine receptors in cockroach ventral nerve cords

1. Effect of 72 ring or α-substituted phenylethanolamines (SPEAs) was examined on the adenylate cyclase prepared from ventral nerve cords of the American cockroach Periplaneta americana.2. Para-Cl-SPEA was the most effective octopaminergic agonist, followed by p-Br-, p-F-, p-Me-, p-NO₂- and p-CF₃-SPEA.3. Meta- and o-SPEAs were less active than p-SPEAs, in stimulating adenylate cyclase.4. SPEA analogs interact with the same binding site as octopamine in the nerve cords of American cockroach, since the level of evoked cAMP production by a combination of optimally effective concentrations of octopamine and SPEA was not greater than the stimulation by octopamine alone.5. Washing removed nearly all of the stimulatory activity of SPEA, suggesting that SPEA binds reversibly to the octopaminergic receptor.     

A wall-bound exo-1,3-β-d-glucanase from Acacia cultured cells

Several wall-bound exo-1,3-β-d-glucanases have been solubilized by 4 M LiCl from suspension-cultured Acacia cells. One exhibits both exo-laminarinase (EC 3.2.1.39) and β-d-glucosidase (EC 3.2.1.21) activities and has been purified up to 30-fold by anion-exchange chromatography, gel filtration and flat-bed electrofocusing. This enzyme hydrolyses laminarin, laminaribiose and p-nitrophenyl-β-d-glucopyranoside. The enzyme, with a pI of 4.6, is apparently homogenous, since it behaves as a single protein with an apparent molecular weight of 62000 on SDS-polyacrylamide gel electrophoresis. Its K_m value in 0.1 M acetate buffer (pH 5.0) with p-nitrophenyl-β-d-glucopyranoside as substrate was 0.27 mM; with laminarin as substrate the K_m expressed in glucosyl residue concentration was 0.64 mM. Other kinetic experiments showed that exo-laminarinase and β-d-glucosidase activities correspond to two distinct catalytic sites in the same protein.     

Transition state stabilization by deaminases: Rates of nonenzymatic hydrolysis of adenosine and cytidine

Nonenzymatic rates of hydrolytic deamination of adenosine and cytidine by acids and bases analogous to side chains of naturally occurring amino acids are compared with the rates of uncatalyzed deamination in water and with the rates of the hydroxide- and hydrogen ion-catalyzed reactions. For adenosine, hydroxide ion is an effective catalyst, with a second-order rate constant of 7.5 × 10⁻⁶ m⁻¹ s⁻¹ at 85°C and an energy of activation of 19.9 kcal/mol. Acid-catalyzed deamination of adenine proceeds with a second-order rate constant of 1.5 × 10⁻⁶ m⁻¹ s⁻¹ at 85°C. At concentrations of 1 m and at pH values corresponding to their respective pK_a values, dimethylamine, acetate, selenide, imidazole, phosphate, and zinc(II) do not enhance the rate of deamination of adenosine beyond that observed in water, and 2-mercaptoethanol produces only a modest rate enhancement. The uncatalyzed rate of adenosine deamination in water is 8.6 × 10⁻⁹ s⁻¹ at 85°C: extrapolation to 37°C and comparison with k_cat for rat hepatoma adenosine deaminase yield a rate enhancement by the enzyme of approximately 2 × 10¹²-fold. 1,6-Dimethyladenosine, the conjugate acid of which has a pK_a value much higher than that of adenosine, is not readily deaminated, suggesting that the uncatalyzed deamination of adenosine does not proceed by hydroxide ion attack on the rare protonated form of adenosine, but rather by attack on the neutral species. Deamination of cytidine is catalyzed most effectively by hydroxide ion, with a second-order rate constant of 4.5 × 10⁻⁴ m⁻¹ s⁻¹ at 85°C and an energy of activation of 28.5 kcal/mol. The uncatalyzed rate of deamination of cytidine in water, which also exhibits an energy of activation of 28.5 kcal/mol, is 8.8 × 10⁻⁸ s⁻¹ at 85°C. Comparison of the rate extrapolated to 25°C with k_cat for bacterial cytidine deaminase gives a rate enhancement for the enzyme of 4 × 10¹¹-fold. The C-5 proton of the pyrimidine ring of cytidine does not exchange with solvent during alkaline hydrolysis, suggesting that deamination under these conditions does not involve prior addition of water across the 5,6 double bond.