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1.
The kinetic α-secondary deuterium isotope effect, , for the pH-independent hydrolysis of nicotinamide riboside, yielding nicotinamide and ribose, in water at 25 ° is 1.14, establishing that this reaction proceeds with unimolecular substrate decomposition to yield a carboxonium ion, or related species, in the rate-determining step. Surprisingly, the corresponding isotope effect for the base-catalyzed decomposition of the same substrate is 1.12, a value indicating considerable sp2 character at the Cl′ position in the transition state for this reaction. A similar result, , was obtained for base-catalyzed hydrolysis of NAD+. The kinetic alpha deuterium isotope effect for the pig brain NAD glycohydrolasecatalyzed hydrolysis of nicotinamide riboside is 1.08. This value suggests that CN bond cleavage to form an intermediate carboxonium ion, or structurally related species, is at least partially rate-determining. In contrast, the corresponding value for the hydrolysis of this substrate catalyzed by Escherichia coli nicotinamide ribonucleotide glycohydrolase is very near unity, a result consistent with several interpretations including a rate-determining enzyme isomerization reaction. 相似文献
2.
N-(2-dimethylaminoethyl)acetohydroxamic acid was synthesized. This compound, which incorporates a dimethylamino group as a second functionality into the hydroxamic acid molecule, catalyzes the hydrolysis of p-nitrophenyl acetate faster than acetohydroxamic acid itself does. The function of the dimethylamino group is to labilize the intermediate formed in the reaction, thus assisting deacylation intramolecularly. The dimethylamino group carries out this function by intramolecular general base catalysis. Nucleophilic catalysis is ruled out by the sizable deuterium oxide solvent isotope effect () found. General acid-hydroxide ion catalysis is ruled out by determination of the lack of reaction with azide ion, which does not possess a dissociable proton, with the intermediate in this reaction. The deuterium oxide solvent isotope effect on the azide ion reaction of the intermediate also rules out a general acid-hydroxide ion reaction. 相似文献
3.
The anomerization of α-d-glucose 6-phosphate has been examined using a spectrophotometric coupled enzyme assay. The pH-rate profile for spontaneous d-glucose 6-phosphate anomerization reveals that the d-glucose 6-phosphate dianion is the species giving rise to the much higher rate of d-glucose 6-phosphate anomerization over that of d-glucose. A deuterium solvent isotope effect of is consistent with the postulated intramolecular general-base catalysis by the phosphate. 相似文献
4.
Leonard M. Hjelmeland Lewis Aronow James R. Trudell 《Biochemical and biophysical research communications》1977,76(2):541-549
Isotope effects for hydroxylation reactions catalyzed by cytochrome P-450 have usually been measured by comparing the overall reaction velocities of deuterated and nondeuterated substrates. Since the rate-limiting step is probably not the single reaction involving covalent bond cleavage, such an approach does not yield information about the primary isotope effect. We measured the primary kinetic isotope effect for benzylic hydroxylation by a method utilizing intramolecular competition, using the symmetrical substrate 1,3-diphenylpropane-1,1-d2. These experiments yield a value of , a larger effect than has previously been reported for benzylic hydroxylations. 相似文献
5.
2-Hydroxymethyl-4-nitrophenyl trimethylacetate is rapidly converted, by an intramolecular pathway, to its benzyl ester counterpart in aqueous solutions of dilute buffers. Intramolecular acyl migration is favored by a factor of 105 over intermolecular transfer of the trimethylacetyl group to surrounding water molecules. The activation parameters of the reaction demonstrate that the rate acceleration is primarily entropic in origin. At constant pH, the apparent first-order rate constant for intramolecular acyl migration displays a linear dependence on the concentration of the basic component of the buffer. For catalysis by imidazole, a solvent deuterium isotope effect of is observed, in accord with a general base-catalyzed pathway. Similarities between intramolecular and intracomplex transacylations are discussed with the conclusion that the migration of a trimethylacetyl group from the phenolic oxygen atom of a 2-hydroxymethyl-4-nitrophenol to the adjacent benzylic oxygen atom provides an accurate model for acylation of the serine hydroxyl group at the active site of α-chymotrypsin by nitrophenyl esters. 相似文献
6.
The kinetics of bisulfite addition to 5-fluorouracil were studied as a function of increasing concentrations of potential general acids. Values of measured at 25°C and ionic strength 1.0 M increased linearly and then became invariant with increasing concentrations of either HSO3? or (OHCH2CH2)2N+C(CH2OH)3 HCl (BisTris+HCl). A small kinetic hydrogen-deuterium isotope effect () was observed for the general acid catalysed portion of the addition reaction. The kinetics of bisulfite elimination from 5-fluoro-5,6-dihydrouracil-6-sulfonate were studied in ethanolamine buffers. As previously observed with 1,3-dimethyl-5,6-dihydrouracil-6-sulfonate, this reaction is subject to general base catalysis and exhibits a large kinetic hydrogen-deuterium isotope effect (). The kinetic results for the addition reaction are consistent with a multistep reaction pathway involving the initial formation of an oxyanion sulfite addition intermediate (II) which subsequently adds a proton and undergoes tautomerization to yield the final 5-fluoro-5,6-dihydrouracil-6-sulfonate product. Thus the elimination of bisulfite from 5-fluoro-5,6-dihydrouracil-6-sulfonate probably proceeds by an ElcB mechanism which involves, at relatively low concentrations of general base, rate determining general base catalyzed proton abstraction from carbon 5 to yield intermediate II followed by the rapid elimination of sulfite to yield 5-fluorouracil. These results may be related to both the enzymatically catalyzed dehalogenation of bromoand iodouracil and the methylation of deoxyuridylate by thymidylate synthetase. 相似文献
7.
The anomerase (1-epimerase) activity of phosphoglucose isomerase (d-glucose 6-phosphate ketol-isomerase EC 5.3.1.9) has been studied. The pH-Vmax profile, assayed by two different methods, shows a dependence on two ionizable groups in the enzyme with pK values of 7.0 and 9.3 at 0 °C. Additionally, an unusual reversal of the basic leg of the normal profile to yield a large increase in Vmax is observed above pH 9.5. Deuterium solvent isotope effects of are observed for isomerase and anomerase activities respectively. An anomerase mechanism similar to noncatalyzed anomerization is postulated with a discussion of the catalytic groups involved. 相似文献
8.
Solvent isotope effects on the structure and function of mitochondrial membranes in aqueous media 总被引:1,自引:0,他引:1
The structural stability of mitochondrial membranes and the enzyme complexes of the electron transport system, and the solubility of a small molecular-weight nonelectrolyte (2-methylnaphthoquinone), have been studied as a function of water structure. D2O, which is considered to be more structured than ordinary water, and H2O were used as solvents in conjunction with chaotropic ions which have been shown to break down water structure. Assays for membrane stability were (a) resolution with respect to solubilization of at least one constituent enzyme, and (b) chaotrope-induced lipid autoxidation, which is a measure of structural destabilization. Solvent isotope effects expressed as the quotient of chaotrope (NaClO4) concentration () necessary to elicit the same effect were found to be (a) essentially constant for each system over a wide range of NaClO4 concentration, and (b) limited to the narrow range of 1.2–1.8 in all tests despite significant differences in the systems studied and the measurements used. The magnitude and the constancy of the isotope effects indicate that increased membrane stability (i.e., the increased strength of hydrophobic interactions in membranes), and decreased water-solubility of nonelectrolytes in D2O are mainly due to the higher degree of order of the deuterated solvent. Thus, in the mitochondrial electron transport chain and many other enzyme systems where solvent isotope effects have been observed, the isotope effect appears to be more a consequence of conformational changes imposed on the enzymes by D2O, because it is a more structured solvent, rather than an indication of direct involvement of protons or the water molecule in the reaction mechanisms. 相似文献
9.
D F Kimball L Peterson D J McLoughlin R G Wolfe 《Archives of biochemistry and biophysics》1979,195(1):66-73
Initial rate, product inhibition, and isotope rate kinetic studies of pig heart mitochondrial and supernatant malate dehydrogenases, acting upon the nonphysiological substrates, meso-tartrate and 2-keto-3-hydroxysuccinate, are reported. The measured spontaneous keto-enol equilibrium for 2-keto-3-hydroxysuccinate in 0.05 m Tris-acetate (pH 8.0) at 25 °C favors the enol form, dihydroxyfumarate, with an apparent equilibrium constant of 0.036. The enzyme-catalyzed reaction favors meso-tartrate with an apparent equilibrium constant of 1.25 × 10?6, M?1 at pH 8.0. The mechanism apparently remains ordered bi bi for both enzymes when these nonphysiological substrates are used, and the chemical-converting hydride transfer step becomes more rate limiting for both enzymes. This conclusion is supported by and values of 2.6 and 3.1, respectively, for the mitochondrial enzyme and 1.9 and 2.9, respectively, for the supernatant enzyme. 相似文献
10.
The rate of reaction of ferro- and ferricytochrome c (C(II) and C(III)) with ferri- and ferrocyanide and of C(III) with O2? and CO2? was determined in H2O and in 2H2O in the temperature range 5–35 °C. No isotope effect was evident in any of the reductions of C(III); the apparent energy of activation was identical in H2O and 2H2O. An isotope effect with , depending on pH for instance was observed in the oxidation of C(II), in the slow phase of oxidation which involves conformational changes. An interpretation (supported by evidence from previous work) involving water molecules in the close vicinity of the reaction site on the protein is discussed. 相似文献
11.
G W Ruddock J A Raleigh C L Greenstock 《Biochemical and biophysical research communications》1981,102(1):554-560
A steady-state competition system has been developed to investigate the reactions of the superoxide radical anion () with various peroxides, including the so-called Haber-Weiss reaction. Potassium superoxide dissolved in an oxygen-free solution of DMSO containing 18-dicyclohexyl-6-crown, is the source of . High pressure liquid chromatography is used as an assay system for reactivity, to detect and quantitate the yield of anthracene, formed as a major product in the reaction between and 9,10-dihydroanthrancene. Decrease in anthracene yields, in the presence of peroxide, may be used to indicate a possible competing reaction between and added peroxide. Complications involving peroxide-stimulated formation of anthraquinone derivatives are discussed. No evidence for a competing reaction between and peroxide can be detected up to a 10-fold excess of peroxide over 9,10-dihydroanthracene. 相似文献
12.
A method is described to measure the oxygen diffusion-concentration product, , at any locus that can be probed or labeled using nitroxide radicals. The method is based on the dependence of the spin-lattice relaxation time of the spin label on the bimolecular collision rate with oxygen. Strong Heisenberg exchange between spin label and oxygen contributes directly to of the spin label, while dipolar interactions are negligible. Both time-domain and continuous wave saturation methods for studying are considered. The method has been applied to phospholipid liposomes using fatty acid spin labels. A discontinuity in at the main phase transition was observed. 相似文献
13.
Richard B. Westkaemper Robert P. Hanzlik 《Archives of biochemistry and biophysics》1981,208(1):195-204
A precise continuous photometric assay has been devised and utilized for mechanistic studies of chicken and rat liver microsomal epoxide hydrolase (EH). The assay is based on monitoring the hydration of p-nitrostyrene oxide (PNSO) at 310 nm. Rat liver EH hydrates S-(+)- and R-(?)-PNSO differentially, the Km and V values for the former being ca. four times those for the latter; in contrast, enantiomeric differences are negligible with chicken liver EH. With rat EH V increases slightly from pH 7 to 8 and then falls rapidly from pH 8 to 9.5; Km remains constant from pH 7 to 8 and then increases steadily from pH 8 to 9.5. In 86 mol% D2O the solvent isotope effect on V () is 1.103 ± 0.015. Both rat and chicken EH show a 3% inverse isotope effect for the hydration of [7-2H]PNSO and a 4% normal isotope effect for the hydration of [8-2H2]PNSO. These observations are discussed in terms of the possible participation of acid as well as base catalysis in the enzymatic mechanism. 相似文献
14.
α-Chymotrypsin, converted to the acetyl enzyme by the p-nitrophenyl esters of CH3COOH, CH2DCOOH, CHD2COOH, and CD3COOH, undergoes deacetylation at pH 7.6 (phosphate buffer) and 25°C with secondary isotope effects of , , and . These isotope effects obey the simple additivity rule (“Rule of the Geometric Mean”) to within 20 J/mol, corresponding to about 5–6% of the maximum isotope effect for carbonyl addition. Thus, to this level, the three hydrogenic sites of the acetyl group are not rendered distinct in their contributions to the overall isotope effect even in the chiral environment of the chymotrypsin active site. 相似文献
15.
The action of xanthine oxidase upon acetaldehyde or xanthine at pH 10.2 has been shown to be accompanied by substantial accumulation of O2? during the first few minutes of the reaction. H2O2 decreases this accumulation of O2? presumably because of the Haber-Weiss reaction () and very small amounts of superoxide dismutase eliminate it. This accumulation of O2? was demonstrated in terms of a burst of reduction of cytochrome , seen when the latter compound was added after aerobic preincubation of xanthine oxidase with its substrate. The kinetic peculiarities of the luminescence seen in the presence of luminol, which previously led to the proposal of H2O4?, can now be satisfactorily explained entirely on the basis of known radical intermediates. 相似文献
16.
Proton inventory investigations of the hydrolysis N-acetylbenzotriazole at pH 3.0 (or the equivalent point on the pD rate profile) have been conducted at two different temperatures and at ionic strengths ranging from 0 to 3.0 M. The solvent deuterium isotope effects and proton inventories are remarkably similar over this wide range of conditions. The proton inventories suggest a cyclic transition state involving four protons contributing to the solvent deuterium isotope effect for the water-catalyzed hydrolysis. The hydrolysis data are described by the equation with , where ko is the observed first-order rate constant in protium oxide, n is the atom fraction of deuterium in the solvent, kn is the rate constant in a protium oxide-deuterium oxide mixture, and is the isotopic fractionation factor. 相似文献
17.
The rate-determining step of the cysteine-catalyzed deiodination of 5-iodouracil is the formation of 5-iodo-6-cysteinyl-5,6-dihydrouracil. The rate of the reaction depends upon the concentration of un-ionized 5-iodouracil and the following ionic species of cysteine; ?OOC(NH3+)CHCH2S?. Unlike the reaction of 2-mercapto-ethanol with 5-iodouracil, the cysteine reaction is not subject to catalysis by imidazolium ion and tris(hydroxymethyl)aminomethane hydrochloride. When the rates of cysteine reacting with 5-iodouracil are measured in both H2O and D2O, a large kinetic isotope effect is observed (), thus implicating the protonated α amino group of cysteine as an intramolecular general acid catalyst for the reaction. These results and possible mechanisms for the actual dehalogenation of the intermediate 5-iodo-6-cysteinyl-5,6-dihydrouracil are discussed in terms of a possible mechanism for enzymatic halopyrimidine dehalogenation. 相似文献
18.
The stoichiometry of free NADPH oxidation in phenobarbital induced rabbit liver microsomes was measured by means of registering the rates of NADPH, H+ and O2 consumption and and H2O2 production. ratio is approximately I indicating that about half H2O2 results from dismutation, the second half being formed directly. ΔNADPH:ΔH2O2 and ΔO2:ΔH2O2 ratios exceed I and therefore another product of the reaction is water. The fact that the ratio (ΔNADPH-ΔH2O2):(ΔO2-ΔH2O2) is 2 allows one to consider direct 4-electron O2 reduction as the major way of water formation rather than endogenous substrate hydroxylation. 相似文献
19.
The reactivities of anionic nitroalkanes with 2-nitropropane dioxygenase of Hansenula mrakii, glucose oxidase of Aspergillus niger, and mammalian d-amino acid oxidase have been compared kinetically. 2-Nitropropane dioxygenase is 1200 and 4800 times more active with anionic 2-nitropropane than d-amino acid oxidase and glucose oxidase, respectively. The apparent Km values for anionic 2-nitropropane are as follows: 2-nitropropane dioxygenase, 1.61 mm; glucose oxidase, 16.7 mm; and d-amino acid oxidase, 11.1 mm. Anionic 2-nitropropane undergoes an oxygenase reaction with 2-nitropropane dioxygenase and glucose oxidase, and an oxidase reaction with d-amino acid oxidase. In contrast, anionic nitroethane is oxidized through an oxygenase reaction by 2-nitropropane dioxygenase, and through an oxidase reaction by glucose oxidase. All nitroalkane oxidations by these three flavoenzymes are inhibited by Cu and Zn-superoxide dismutase of bovine blood, Mn-superoxide dismutases of bacilli, Fe-superoxide dismutase of Serratia marcescens, and other scavengers such as cytochrome c and NADH, but are not affected by hydroxyl radical scavengers such as mannitol. None of the scavengers tested affected the inherent substrate oxidation by glucose oxidase and d-amino acid oxidase. Furthermore, the generation of in the oxidation of anionic 2-nitropropane by 2-nitropropane dioxygenase was revealed by ESR spectroscoy. The ESR spectrum of anionic 2-nitropropane plus 2-nitropropane dioxygenase shows signals at g1 = 2.007 and g11 = 2.051, which are characteristic of . The generated is a catalytically essential intermediate in the oxidation of anionic nitroalkanes by the enzymes. 相似文献
20.
Three monofluorocinnamoylchymotrypsins have been examined at pH 4 by fluorine NMR spectroscopy. Protein-induced fluorine chemical shifts are quite large (~7 ppm) when fluorine is present at the para position but nearly zero for ortho fluorine. The shifts roughly parallel those observed in complexes formed between the enzyme and the analogous N-acetylfluorophenylalanines, suggesting a similarity in molecular environment for the aromatic ring in both systems. Little correlation is found, however, between the shifts for the acylenzymes and those of the corresponding enzyme-cinnamate complexes, indicating that the environment for the aromatic ring in the complexes is dissimilar from that experienced by the aromatic group in the acylated enzyme. Solvent isotope effects () on the fluorine chemical shifts for the fluorocinnamoylchymotrypsins are small and downfield. Fluorine NMR observations suggest that the presence of the fluorocinnamoyl group greatly stabilizes the enzyme toward denaturation in 8 m urea. 相似文献