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At least 73 records · Page 4Linked to original sources
PubMed1998

Pre-steady-state analysis of ATP hydrolysis by Saccharomyces cerevisiae DNA topoisomerase II. 1. A DNA-dependent burst in ATP hydrolysis.

When bound to DNA, topoisomerase II from Saccharomyces cerevisiae exhibits burst kinetics with respect to ATP hydrolysis. Pre-steady-state analysis shows that the enzyme binds and hydrolyzes two ATP per reaction cycle. Our data indicate that at least one of the two ATP is rapidly hydrolyzed prior to the rate-determining step in the reaction mechanism. When DNA is not bound to topoisomerase II, the rate-determining step shifts to become either ATP binding or hydrolysis. Two possible mechanisms are proposed that agree with our observations.

Adenosine Triphosphate↗
PubMed2001

Solvent effects on the reaction coordinate of the hydrolysis of phosphates and sulfates: application of Hammond and anti-Hammond postulates to understand hydrolysis in solution.

The mechanism of the alkaline hydrolysis of phosphate and sulfate esters is of great interest. Ab initio quantum mechanical calculations and dielectric continuum methods are used to investigate the effect of the solvent on the associative/dissociative and the in-line/sideways character of the hydrolysis reaction of ethylene sulfate (ES) and ethylene phosphate (EP(-)), and their acyclic counterparts, dimethyl sulfate (DMS) and dimethyl phosphate (DMP(-)). The gas-phase reaction coordinates are determined by Hartree-Fock and density functional theory. For ES, the reaction coordinate in solution is determined; for the other three reactions only the transition state in solution is obtained. The alterations in the reaction induced by solvent are interpreted by use of the Hammond and anti-Hammond postulates.

Ethylenes↗
PubMed1999

Protein hydrolysis and proteinase activity during the ripening of salted anchovy (Engraulis encrasicholus l.). A microassay method for determining the protein hydrolysis.

The protein hydrolysis and proteinase activity during the ripening of salted anchovy were studied. A rapid, simple, and inexpensive microassay method for determining the protein hydrolysis by trinitrobenzenesulfonic acid (TNBS) has been developed. A linear relationship was observed between proteolysis determination by the TNBS method and ripening time in the fish muscle and in the brine (r = 0.99). A linear relationship was also observed between the ratio nonprotein nitrogen and total nitrogen (NPN/TN) and ripening time (r = 0.98). Proteolysis by the TNBS method and NPN/TN determination could be considered as objective methods to follow and assess the ripening process of an anchovy. A value of proteolysis by the TNBS method of 240 mM leucine in the fish muscle and/or 200 mM leucine in the brine would indicate the ripening point. The crude enzyme prepared of fish muscle and brine showed that alkaline proteinases dominate.

Animals↗
PubMed2002

Intramolecular general base catalyzed ester hydrolysis. The hydrolysis of 2-aminobenzoate esters.

Rate constants have been obtained for the hydrolysis of the trifluoroethyl, phenyl, and p-nitrophenyl esters of 2-aminobenzoic acid at 50 degrees C in H(2)O. The pseudo-first-order rate constants, k(obsd), are pH independent from pH 8 to pH 4 (the pK(a) of the amine group conjugate acid). The 2-aminobenzoate esters hydrolyze with similar rate constants in the pH-independent reactions, and these water reactions are approximately 2-fold slower in D(2)O than in H(2)O. The most likely mechanism involves intramolecular general base catalysis by the neighboring amine group. The rate enhancements in the pH-independent reaction in comparison with the pH-independent hydrolysis of the corresponding para substituted esters or the benzoate esters are 50-100-fold. In comparison with the hydroxide ion catalyzed reaction, the enhancement in k(obsd) at pH 4 with the phenyl ester is 10(5)-fold. Intramolecular general base catalyzed reactions are assessed in respect to their relative advantages and disadvantages in enzyme catalysis. A general base catalyzed reaction can be more rapid at low pH than a nucleophilic reaction that has a marked dependence on pH and the leaving group.

Catalysis↗
PubMed1965

PAPAIN-CATALYSED HYDROLYSIS OF SOME HIPPURIC ESTERS. A NEW MECHANISM FOR PAPAIN-CATALYSED HYDROLYSIS.

1. The Michaelis-Menten parameters for the papain-catalysed hydrolysis of a number of alkyl, aryl and alkyl-thiol esters of hippuric acid have been determined. 2. For all the aryl esters and most of the alkyl esters studied, the catalytic constant, k(0), is 2-3sec.(-1) and most probably represents deacylation of the common intermediate, hippuryl-papain. 3. Two alkyl esters and hippurylamide, however, have catalytic rate constants, k(0), less than 2-3sec.(-1). It is possible to interpret all the available kinetic data in terms of a three-step mechanism in which an enzyme-substrate complex is first formed, followed by acylation of the enzyme through an essential thiol group, followed by deacylation of the acyl-enzyme. 4. The logarithm of the ratio of the Michaelis-Menten parameters, which reflect the acylation rate constant, for four aryl esters of hippuric acid studied give a linear Hammett plot against the substituent constant, sigma. Arguments are presented that indicate acid as well as nucleophilic catalysis in the acylation process and that the most likely proton donor is an imidazolium ion. 5. It is suggested that this imidazolium ion is part of the same histidine residue that has been tentatively implicated in the deacylation process (Lowe & Williams, 1965b). 6. A new mechanism is proposed for the papain-catalysed hydrolysis of N-acyl-alpha-amino acid derivatives.

Acylation↗
PubMed1994

Cellulose hydrolysis by the cellulases from Trichoderma reesei: adsorptions of two cellobiohydrolases, two endocellulases and their core proteins on filter paper and their relation to hydrolysis.

Separate binding of several purified cellulolytic components of Trichoderma reesei on to filter paper was studied and concomitant hydrolysis rates evaluated. Enhancement of mass transfer from the bulk liquid to the solid substrate by agitation has two different effects on adsorption depending on the type of enzyme: (i) the fraction of cellobiohydrolase II (CBH II) and endoglucanase III (EG III) bound at equilibrium is increased, whereas (ii) the rate but not the extent of cellobiohydrolase I (CBH I) and endoglucanase I (EG I) adsorption is affected. The adsorption of CBH I core, a component lacking the cellulose-binding domain (CBD), is, however, not significantly influenced by mass transfer. The CBH I interdomain peptide (present in CBH I core b) does not participate in adsorption but enhances stability. The adsorption of CBH I core proteins is a fully reversible process whereas that of the intact CBH I is not. Thus, the interaction of the CBD with filter paper apparently accounts for the mass-transfer-limited binding rate and also for the irreversible adsorption of intact CBH I. Adsorption isotherms at 50 degrees C indicate very similar relative association constants for the intact cellulases (0.24-0.30 l/g of cellulose), but drastically reduced values for CBH I core proteins (0.03 l/g of cellulose). The specific activities of adsorbed CBH I and of its core proteins are identical and a linear relationship between adsorption and rates of hydrolysis is found only for these enzymes. Thus, non-productive binding on to cellulose seems evident in the case of CBH II and EG III but not CBH I.

Adsorption↗
PubMed1996

Biochemical analysis of mutant T7 primase/helicase proteins defective in DNA binding, nucleotide hydrolysis, and the coupling of hydrolysis with DNA unwinding.

We characterized nine helicase-deficient mutants of bacteriophage T7 helicase-primase protein (4A') prepared by random mutagenesis as reported in the accompanying paper (Rosenberg, A. H., Griffin, K., Washington, M. T., Patel, S. S., and Studier, F. W. (1996) J. Biol. Chem. 271, 26819-26824). Mutants were selected from each of the helicase-conserved motifs for detailed analysis to understand better their function. In agreement with the in vivo results, the mutants were defective in helicase activity but were active in primase function. dTTP hydrolysis, DNA binding, and hexamer formation were examined. Three classes of defective mutants were observed. Group A mutants (E348K, D424N, and S496F), defective in dTTP hydrolysis, lie in motifs 1a, 2, and 4 and are possibly involved in NTP binding/hydrolysis. Group B mutants (R487C and G488D), defective in DNA binding, lie in motif 4 and are responsible directly or indirectly for DNA binding. Group C mutants (G116D, A257T, S345F, and G451E) were not defective in any of the activities except the helicase function. These mutants, scattered throughout the protein, appear defective in coupling dTTPase activity to helicase function. Secondary structural predictions of 4A' and DnaB helicases resemble the known structures of RecA and F1-ATPase enzymes. Alignment shows a striking correlation in the positions of the amino acids that interact with NTP and DNA.

Amino Acid Sequence↗
PubMedDate not supplied

The nonenzymatic hydrolysis of oligoribonucleotides. VII. Structural elements affecting hydrolysis.

Several elements of oligoribonucleotide structure are important for efficient hydrolysis. We have found that the following factors influence oligoribonucleotide hydrolysis: (i) single-stranded structure of RNA flanking the scissile phosphodiester bond, (ii) the substituent on atom C-5 of the uridine adjacent to the cleaved internucleotide bond, (iii) the position of the scissile UA phosphodiester bond within a hairpin loop, (iv) the concentration of formamide, urea, ethanol and sodium chloride.

Chromatography, High Pressure Liquid↗
PubMed1991

[Hydrolysis of raw keratin wastes with use of certain sulfonic acids. II. Analysis of optimal conditions for hydrolysis].

We have fixed optimal conditions to hydrolysis of the waste keratin material cerebellum, hoof and bristle. With apply 1 mol/dm3 solutions of acids: amide-, benzene-, 4-methylbenzene- and naphtalene-1-sulfonate, the best results obtained by degradation protein for 24 hours in closed glass tube in the argon in the temperature 110 degrees C by means of benzenesulfonate acid; materials after hydrolysis contain all exogenous amino acids.

Animals↗
PubMed1997

Homogeneous catalyst for DNA hydrolysis (4). Efficient DNA hydrolysis by lanthanide--saccharide complexes.

Homogeneous and neutral solutions are prepared by mixing Ce(NH4)2(NO3)6 with either isomaltose, melibiose, gentiobiose, palatinose, mannitol, sorbitol, galactitol, or glucamine in pH 7 hepes buffer ([Ce(IV)]0/[monomeric residue of saccharide]0 = 1). In contrast, amylose, cyclodextrins, maltose, glucose, and fructose provide only heterogeneous mixtures. The homogeneous solution of 1:1 Ce(IV)/glucamine system is active for DNA hydrolysis: the pseudo-first-order rate constant for the hydrolysis of thymidylyl(3'-->5')thymidine at pH 7.0 and 50 degrees C is 0.010 h-1, when [Ce(IV)]0 = [glucamine]0 = 10 mmol dm-3. The DNA-hydrolyzing activity decreases in the following order: glucamine > isomaltose, melibiose, gentiobiose >> palatinose, mannitol, sorbitol, galactitol.

Catalysis↗
PubMed1998

Rapid hydrolysis of bile acid conjugates using microwaves: retention of absolute stereochemistry in the hydrolysis of (25R) 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oyltaurine.

In recent years, defects of bile acid synthesis caused by disorders of peroxisome biogenesis have led to increased interest in C27 bile acids. In humans, while the majority of bile acids are C24 carboxylic acids, the presence of increased concentrations of C27 bile acids and their metabolites in hereditary diseases associated with peroxisomal dysfunction can serve as a useful marker for the intensity of the metabolic disorder. Our present studies describe an efficient method for the rapid hydrolysis of C27 and C24 bile acid conjugates using a commercial microwave oven. The advantages of this method include freedom from racemization, minimal activation, mild reaction conditions, and the highly stereocontrolled nature of the reaction, thus allowing for free bile acid recovery in high yield. For example, when (25R) 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oyl taurine, a major compound present in the bile of Alligator mississippiensis, was deconjugated with 4% NaOH/diethylene glycol or 1 M LiOH/propylene glycol in the microwave oven for 4-6 min, 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid (THCA) was obtained in 81% yield with retention of configuration at C-25. It is suggested that present studies will be helpful in delineating the absolute stereochemistry of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoyl-CoA oxidase, the peroxisomal enzyme that catalyzes the first step in the oxidation of THCA.

Alligators and Crocodiles↗
PubMed1985

Stereoselective hydrolysis of 16 alpha-halo-17-keto steroids and long-range substitution effects on the hydrolysis of 16 alpha-bromo-17-ketones and 2 alpha-bromo-3-ketones.

Epimerizations of 16 alpha-chloro- (1a), bromo- (1b), and iodo-3 beta-hydroxy-5-androsten-17-one (1c) by a brief treatment with 0.2 equiv NaOH in aqueous pyridine reached equilibrium between 16 alpha- and 16 beta-halo ketones. 16 alpha-/16 beta-Halo ketone ratios at equilibrium were 1.5 for Cl, 1.25 for Br, and 1.0 for I. Kinetic analysis showed that compounds 1a-c were stereoselectively converted to the corresponding 16 alpha-hydroxy derivative 3 by an SN2 mechanism, in which the order of the apparent reactivity was Br greater than I greater than Cl. The hydrolysis of a number of 16 alpha-bromo-17-ketones and 2 alpha-bromo-3-ketones was carried out. The yields of the corresponding alcohols were found to depend on remote structural features in the steroids.

17-Ketosteroids↗
PubMed1984

Actinidin hydrolysis of substituted-phenyl hippurates: a quantitative structure-activity relationship and graphics comparison with hydrolysis by papain.

The hydrolysis of 29 phenyl hippurates (XPhOCOCH2NHC(=O)C6H5) by the cysteine protease actinidin has been studied and a quantitative structure-activity relationship (QSAR) has been formulated: log 1/Km = 0.74 sigma + 0.50 pi'3 + 0.24MR4 + 2.90. In this expression Km is the Michaelis constant, sigma is the Hammett constant, pi'3 is the hydrophobic parameter for the more hydrophobic of the two meta substituents, and MR4 is the molar refractivity of para substituents. The QSAR for actinidin is compared with a similar one obtained for another cysteine plant protease papain. A color stereo computer graphics model constructed from the X-ray crystallographic coordinates of actinidin is compared with those of our previously reported models for papain.

Cysteine Endopeptidases↗
PubMedDate not supplied

[Coupling of proteolysis and hydrolysis of ATP upon functioning of Lon proteinase of Escherichia coli. II. Hydrolysis of ATP and activity of peptide hydrolase sites of the enzyme].

The absence of direct correlation between the efficiency of functioning of ATPase and peptidehydrolase sites of Lon protease was revealed. It was shown that Lon protease is an allosteric enzyme, in which the catalytic activity of peptidehydrolase sites is determined by the binding of nucleotides, their magnesium complexes, and free magnesium ions in the enzyme's ATPase sites. It was revealed that complex ADP-Mg, an inhibitor of the native enzyme, is an activator of the Lon-K362Q form of the Lon protease mutant in the ATPase site. Considered are variants of intersite functional contacts realizing in the enzyme. The existence of two ways of signal transduction was established from the ATPase sites to peptidehydrolase ones in the Lon protease oligomer--intra- and intersubunit ways. Location of the enzyme ATPase sites is suggested in the areas of the complementary surfaces of subunits. It is hypothesized that ATP hydrolysis upon degradation of protein substrates by the E. coli Lon protease in vivo acts as a factor of restriction of the enzyme's degrading activity.

ATP-Dependent Proteases↗
PubMed1997

Biochemical analyses of mutations in the HSV-1 helicase-primase that alter ATP hydrolysis, DNA unwinding, and coupling between hydrolysis and unwinding.

Herpes simplex virus type 1 encodes a heterotrimeric helicase-primase composed of the products of the UL5, UL52, and UL8 genes. UL5 possesses six motifs conserved among superfamily 1 of helicase proteins. Substitutions of conserved residues in each motif abolishes DNA replication in vivo (Zhu, L., and Weller, S. K. (1992) J. Virol. 66, 469-479). Purified UL5.52 harboring a Gly to Ala change in motif V retains primase and helicase activities in vitro but exhibits a higher KM for single-stranded DNA and lower DNA-dependent ATPase activity (Graves-Woodward, K. L., and Weller, S. K. (1996) J. Biol. Chem. 272, 13629-13635). We have purified and characterized six other subcomplexes with residue changes in the UL5 helicase motifs. Each variant subcomplex displays at least wild type or greater levels of primase and DNA binding activities, but all are defective in helicase activity. Mutations in motifs I and II exhibit profound decreases in DNA-dependent ATPase activity. Mutations in motifs III-VI decrease DNA-dependent ATPase activity 3-6-fold. Since mutations in motifs III, IV, V, and VI do not eliminate ATP hydrolysis or DNA binding, we propose that they may be involved in the coupling of these two activities to the process of DNA unwinding. This analysis represents the first comprehensive structure-function analysis of the conserved motifs in helicase superfamily 1.

Adenosine Triphosphatases↗
PubMed2004

Combination of S1 nuclease and PNA for site-selective hydrolysis of double-stranded DNA. Comparison with the site-selective hydrolysis using Ce(IV)/EDTA.

The potential of the combination of SI nuclease and pseudo-complementary PNA (pcPNA) for site-selective scission of double-stranded DNA has been investigated. Through strand invasion of two pcPNAs, single-stranded portions were formed in both strands of substrate DNA. In the initial stage of the enzymatic digestion, two scission fragments were obtained due to the hydrolysis at these two gap-like sites. On prolonged reactions, however, these products (as well as the substrate DNA) were further digested to smaller fragments. Under the conditions employed here, only Ce(IV)/EDTA is available for the preparation of desired fragments from double-stranded DNA.

Cerium↗
PubMed2000

[Kinetics of hydrolysis of acylderivatives of stobadine, prodrug forms of free oxygen radical scavengers. Part 2: alkaline hydrolysis].

Stobadin, (-)-cis-2,8-dimethyl-2,3,4,4a,5,9b-hexahydro-1H-pyridol[4,3b]- indol is a compound with a potential antiarrhythmic, antihistamine, anaesthetic, antiulcerous, and marked antioxidative effect. N5-acylsubstitution of stobadin yielded derivatives, which represent prodrug forms of the active principle--stobadin, and it is assumed that they will be hydrolysed in the organism and the active principle will be released in higher concentrations in various biological tissues. The present paper deals with the investigation of the kinetics of hydrolysis of 13 stobadin acylderivatives in a medium of 0.1 mol/l of sodium hydroxide at 70 degrees C, employing spectrophotometry in the UV region of the spectrum. The determined rate constants were correlated with the length of the side acyl chain and the pKa values of the compounds under study.

Carbolines↗
PubMed2001

[Hydrolysis kinetics of stobadine acyl-derivatives, the prodrug forms of free oxygen radical extinguishers. Part 3. Hydrolysis in a neutral medium].

The pyridoindole derivative stobadin, [(-)-cis-2,8-dimetyl-2,3,4,4a,5,9b-hexahydro-1H-pyrido[4,3b]-indole] is a perspective antiarrhythmic, antihistamine, anaesthetic, antiulcerous drug capable of extinguishing free oxygen radical. Its prodrug forms--N(5)- acyl-substituted stobadine--of the active substance--stobadine--have been prepared and it is assumed that the will be hydrolyzed in the organism and the active substance will be released in higher concentrations in different biological tissues. The present paper is concerned with the investigation of the kinetics of the hydrolysis of 13 acyl derivatives of stobadine in the medium of a buffer solution of pH 7 at temperatures of 70 degrees C and 75 degrees C spectrophotometrically in the UV region of the spectrum. The determined rate constants were correlated with the length of the side acyl chain and the pKa values of the drugs under study. The profile of log k--pH of substances was determined.

Carbolines↗