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K Brocklehurst

Publications and source records attributed to K Brocklehurst.

At least 145 records · Page 8Linked to original sources
PubMed1976

4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole as a reactivity probe for the investigation of the thiol proteinases. evidence that ficin and bromelain may lack carboxyl groups conformationally equivalent to that of aspartic acid-158 of papain.

1. 4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole (Nbd chloride) was used as a reactivity probe to characterize the active centres of papin (EC 3.4.22.2), ficin (EC 3.4.22.3) and bromelain (EC 3.4.22.4). 2. In the pH range 0-8 Nbd chloride probably exists mainly as a monocation, possibly with the proton located on N-1 of the oxadiazole ring. 3. Spectroscopic evidence is presented for the intermediacy of Meisenheimer-type adducts in the reaction of Nbd chloride with nucleophiles. 4. The pH-dependence of the second-order rate constants (k) of the reactions of the three enzymes with Nbd chloride was determined at 25 degrees C, I = 0.1 mol/litre in 6.7% (v/v) ethanol in the pH range 2.5-5, where, at least for papain and ficin, the reactions occur specifically with their active-centre thiol groups. The pH-k profile for the papain reaction is bell-shaped (pKaI = 3.24, pKaII = 3.44 and k = 86M(-1)-s(-1), whereas that for ficin is sigmoidal (pKa = 3.6, k = 0.36M(-1)-s(-1), the rate increasing with increasing pH. The profile for the bromelain reaction appears to resemble that for the ficin reaction, but is complicated by amino-group labelling. 5. The bell-shaped profile of the papain reaction is considered to arise from the reaction of the thiolate ion of cysteine-25, maintained in acidic media by interaction with the side chain of histidine-159, with the Nbd chloride monocation hydrogen-bonded at its nitro group to the un-ionized form of the carboxyl group of aspartic acid-158. The lack of acid catalysis in the corresponding reactions of ficin and probably of bromelain suggests that these enzymes may lack carboxyl groups conformationally equivalent to that of aspartic acid-158 of papain. The possible consequences of this for the catalytic sites of these enzymes is discussed.

4-Chloro-7-nitrobenzofurazan↗
PubMed1976

A convenient method of preparation of high-activity urease from Canavalia ensiformis by covalent chromatography and an investigation of its thiol groups with 2,2'-dipyridyl disulphide as a thiol titrant and reactivity probe.

1. A convenient method of preparation of jack-bean urease (EC3.5.1.5) involving covalent chromatography by thiol-disulphide interchange is described. 2. Urease thus prepared has specific activity comparable with the highest value yet reported (44.5 +/- 1.47 kat/kg, Km = 3.32 +/- 0.05 mM; kcat. = 2.15 X 10(4) +/- 0.05 X 10(4)s-1 at pH7.0 and 38 degrees C). 3. Titration of the urease thiol groups with 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py) and application of the method of Tsou Chen-Lu [(1962) Sci. Sin. 11, 1535-1558] suggests that the urease molecule (assumed to have mol.wt. 483000 and epsilon280 = 2.84 X 10(5) litre-mol-1-cm-1) contains 24 inessential thiol groups of relatively high reactivity (class-I), six 'essential' thiol groups of low reactivity (class-II) and 54 buried thiol groups (class-III) which are exposed in 6M-guanidinium chloride. 4. The reaction of the class-I thiol groups with 2-Py-S-S-2-Py was studied in the pH range 6-11 at 25 degrees C(I = 0.1 mol/l) by stopped-flow spectrophotometry, and the analogous reaction of the class-II thiol groups by conventional spectrophotometry. 5. The class-I thiol groups consist of at least two sub-classes whose reactions with 2-Py-S-S-2-Py are characterized by (a) pKa = 9.1, k = 1.56 X 10(4)M-1-s-1 and (b) pKa = 8.1, k = 8.05 X 10(2)M-1-s-1 respectively. The reaction of the class-II thiol groups is characterized by pKa = 9.15 and k = 1.60 X 10(2)M-1-s-1. 6. At pH values 7-8 the class-I thiol groups consist of approx. 50% class-Ia groups and 50% class-Ib groups. The ratio class Ia/class Ib decreases an or equal to approx. 9.5, and at high pH the class-I thiol groups consist of at most 25% class-Ia groups and at least 75% class-Ib groups. 7. The reactivity of the class-II thiol groups towards 2-Py-S-S-2-Py is insensitive to the nature of the group used to block the class-I thiols. 8. All the 'essential' thiol groups in urease appear to be eeactive only as uncomplicated thiolate ions. The implications of this for the active-centre chemistry of urease relative to that of the thiol proteinases are discussed.

2,2'-Dipyridyl↗
PubMed1975

A reporter group delivery system with both absolute and selective specificity for thiol groups and an improved fluorescent probe containing the 7-nitrobenzo-2-oxa-1,3-diazole moiety.

1. 4-(N-2-Aminoethyl2'-pyridyl disulphide)-7-nitrobenzo-2-oxa-1,3-diazole (compound I) was synthesized and evaluated as a fluorescent labelling reagent for thiol groups. 2. The design of compound (I) as one example of a general type of reporter group delivery reagent (2-pyridyl-S-S-X, where X contains an environmentally sensitive spectroscopic probe) is discussed. 3. The electronic absorption spectrum of compound (I) was determined over a wide range of pH and the spectral changes that accompany its reaction with low-molecular-weight thiols, e.g. L-cysteine, and with papain (EC 3.4.22.2) and bovine serum albumin are discussed. 4. A new value of epsilon343 for 2-thiopyridone (Py-2-SH) was determined as 8.08 X 10(3) +/- 0.08 X 10(3)M-1-cm-1. 5. Spectral analysis of the reactions of compound (I) with L-cysteine and with papain (in the pH range 3.5-8.0) showed that even under equimolar conditions the reaction (thiol-disulphide interchange to release Py-2-SH) is essentially stoicheimoetric and probably proceeds by specific attack at the sulphur atom distal from the pyridyl ring of compound (I). 6. The fluorescence-emission spectra of compound (I) and of the products of its reaction with papain and with ficin (EC 3.4.22.3) were determined. Compound (I) is highly fluorescent in aqueous solution. Excitation within the intense visible absorption band (lambda max. 481 nm, epsilon max. 2.52 X 10(4)M-1-cm-1) provides green fluorescence with an emission maximum at 540 nm. Both papain and ficin labelled by reaction with compound (I) are characterized by fluorescence-emission maxima (535 nm and 530 nm respectively) of even higher intensity. The fluorescence emission of the product of the reaction of papain with compound (I) was shown to be 25 times more intense than that of the product of the reaction of papain with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (Nbd chloride). 7. The second-order rate constants (k2) for the reactions of compound (I) and of Nbd chloride with GSH, papain, albumin, ficin, 2-benzimidazolylmethanethiol and 2-benzimidazolylethanethiol were determined at 25.0 degrees C and various pH values. At pH4 the values of k2(compound I)/k2(Nbd chloride) are: GSH, 288; albumin, 36; papain 3 X 10(3); ficin, 3 X 10(4). 8. The pH-k2 profiles for the reactions of compound (I) and of Nbd chloride with the two 2-benzimidazolylalkanethiols were determined. Of the four profiles only that for the reaction of compound (I) with 2-benzimidazolylmethanethiol is characterized by a striking rate maximum in acidic media.

Benzimidazoles↗
PubMed1974

Reactions of L-ergothioneine and some other aminothiones with2,2'-and 4,4'-dipyridyl disulphides and of L-ergothioneine with iodoacetamide. 2-Mercaptoimidazoles, 2- and 4-thiopyridones, thiourea and thioacetamide as highly reactive neutral sulphur nucleophils.

1. The reactions of 2,2'- and 4,4'-dipyridyl disulphide (2-Py-S-S-2-Py and 4-Py-S-S-4-Py) with l-ergothioneine (2-mercapto-l-histidine betaine), 2-mercaptoimidazole, 1-methyl-2-mercaptoimidazole, thiourea, thioacetamide, 2-thiopyridone (Py-2-SH) and 4-thiopyridone (Py-4-SH) were investigated spectrophotometrically in the pH range approx. 1-9. 2. These reactions involve two sequential reversible thiol-disulphide interchanges. 3. The reaction of l-ergothioneine with 2-Py-S-S-2-Py and/or with the l-ergothioneine-Py-2-SH mixed disulphide, both of which provide Py-2-SH, is characterized by at least three reactive protonic states. This provides definitive evidence that neutral l-ergothioneine is a reactive nucleophile, particularly towards the highly electrophilic protonated disulphides. 4. A similar situation appears to obtain in the reactions of l-ergothioneine and Py-2-SH with 4-Py-S-S-4-Py and in the reactions of the other 2-mercaptoimidazoles, thiourea and Py-4-SH with 2-Py-S-S-2-Py. The nucleophilic reactivity of Py-4-SH suggests that general base catalysis provided by the disulphide in a cyclic or quasi-cyclic transition state is not necessary to generate nucleophilic reactivity in the other amino-thiones whose geometry could permit such catalysis. 5. The existence of a positive deuterium isotope effect in the l-ergothioneine-2-Py-S-S-2-Py system at pH6-7 provides no evidence for general base catalysis but is in accord with a mechanism involving specific acid catalysis and post-transition-state proton transfer. 6. The pH-dependences of the overall equilibrium positions of the various thiol-disulphide interchanges are described. 7. Reaction of thioacetamide with a stoicheiometric quantity of 2-Py-S-S-2-Py at pH1 provides 2 molecules of Py-2-SH per molecule of thioacetamide and elemental sulphur; these findings can be accounted for by thiol-disulphide interchange to provide a thioacetamide-Py-2-SH mixed disulphide followed by fragmentation to provide CH(3)CN, S and Py-2-SH. 8. Provision of high reactivity in the neutral forms of the members of this series of sulphur nucleophiles by electron donation by the amino group is compared with the well known alpha effect that provides enhanced nucleophilicity in compounds containing an electronegative atom adjacent to the nucleophilic atom. 9. The decrease in the u.v. absorption of l-ergothioneine at 257nm consequent on transformation of its aminothione moiety into an S-alkyl-2-mercaptoimidazole moiety provides a convenient method of following the alkylation of l-ergothioneine by iodoacetamide. 10. The pH dependence of the extinction coefficient of l-ergothioneine at 257nm is described by epsilon(257)={8x10(3)/(1+K(a)/[H(+)]} +6x10(3)m(-1).cm(-1) in which pK(a)=10.8. 11. In the pH range 3-11 the reaction is characterized by two reactive protonic states (X and XH). 12. The X state, reaction of the ionized 2-mercaptoimidazole moiety of the l-ergothioneine dianion with neutral iodoacetamide, is characterized by the second-order rate constant 4.0m(-1).s(-1) (25.0 degrees C, I=0.05). The XH state, characterized by the second-order rate constant 0.03m(-1).s(-1), is interpreted as reaction of the thione form of the neutral 2-mercaptoimidazole moiety of the l-ergothioneine monoanion with neutral iodoacetamide. 13. The XH state of the alkylation reaction does not exhibit a deuterium isotope effect.

2,2'-Dipyridyl↗
PubMed1974

A convenient spectrophotometric assay for the determination of L-ergothioneine in blood.

1. A convenient spectrophotometric assay for the determination of l-ergothioneine in solution including deproteinized blood haemolysate was developed. 2. The method consists of deproteinization by heat precipitation and Cu(2+)-catalysed oxidation of thiols such as glutathione and of l-ascorbic acid, both in alkaline media, and titration of l-ergothioneine (which is not oxidized under these conditions) by its virtually instantaneous reaction with 2,2'-dipyridyl disulphide at pH1. 3. This method and the results obtained with it for the analysis of human, horse, sheep and pig blood are compared with existing methods of l-ergothioneine analysis and the results obtained thereby.

Animals↗
PubMed1974

Kinetics of the hydrolysis of N-benzoyl-L-serine methyl ester catalysed by bromelain and by papain. Analysis of modifier mechanisms by lattice nomography, computational methods of parameter evaluation for substrate-activated catalyses and consequences of postulated non-productive binding in bromelain- and papain-catalysed hydrolyses.

1. N-Benzoyl-l-serine methyl ester was synthesized and evaluated as a substrate for bromelain (EC 3.4.22.4) and for papain (EC 3.4.22.2). 2. For the bromelain-catalysed hydrolysis at pH7.0, plots of [S(0)]/v(i) (initial substrate concn./initial velocity) versus [S(0)] are markedly curved, concave downwards. 3. Analysis by lattice nomography of a modifier kinetic mechanism in which the modifier is substrate reveals that concave-down [S(0)]/v(i) versus [S(0)] plots can arise when the ratio of the rate constants that characterize the breakdown of the binary (ES) and ternary (SES) complexes is either less than or greater than 1. In the latter case, there are severe restrictions on the values that may be taken by the ratio of the dissociation constants of the productive and non-productive binary complexes. 4. Concave-down [S(0)]/v(i) versus [S(0)] plots cannot arise from compulsory substrate activation. 5. Computational methods, based on function minimization, for determination of the apparent parameters that characterize a non-compulsory substrate-activated catalysis are described. 6. In an attempt to interpret the catalysis by bromelain of the hydrolysis of N-benzoyl-l-serine methyl ester in terms of substrate activation, the general substrate-activation model was simplified to one in which only one binary ES complex (that which gives rise directly to products) can form. 7. In terms of this model, the bromelain-catalysed hydrolysis of N-benzoyl-l-serine methyl ester at pH7.0, I=0.1 and 25 degrees C is characterized by K(m) (1) (the dissociation constant of ES)=1.22+/-0.73mm, k (the rate constant for the breakdown of ES to E+products, P)=1.57x10(-2)+/-0.32x10(-2)s(-1), K(a) (2) (the dissociation constant that characterizes the breakdown of SES to ES and S)=0.38+/-0.06m, and k' (the rate constant for the breakdown of SES to E+P+S)=0.45+/-0.04s(-1). 8. These parameters are compared with those in the literature that characterize the bromelain-catalysed hydrolysis of alpha-N-benzoyl-l-arginine ethyl ester and of alpha-N-benzoyl-l-arginine amide; K(m) (1) and k for the serine ester hydrolysis are somewhat similar to K(m) and k(cat.) for the arginine amide hydrolysis and K(as) and k' for the serine ester hydrolysis are somewhat similar to K(m) and k(cat.) for the arginine ester hydrolysis. 9. A previous interpretation of the inter-relationships of the values of k(cat.) and K(m) for the bromelain-catalysed hydrolysis of the arginine ester and amide substrates is discussed critically and an alternative interpretation involving substantial non-productive binding of the arginine amide substrate to bromelain is suggested. 10. The parameters for the bromelain-catalysed hydrolysis of the serine ester substrate are tentatively interpreted in terms of non-productive binding in the binary complex and a decrease of this type of binding by ternary complex-formation. 11. The Michaelis parameters for the papain-catalysed hydrolysis of the serine ester substrate (K(m)=52+/-4mm, k(cat.)=2.80+/-0.1s(-1) at pH7.0, I=0.1, 25.0 degrees C) are similar to those for the papain-catalysed hydrolysis of methyl hippurate. 12. Urea and guanidine hydrochloride at concentrations of 1m have only small effects on the kinetic parameters for the hydrolysis of the serine ester substrate catalysed by bromelain and by papain.

Binding Sites↗
PubMed1973

Reactions of papain and of low-molecular-weight thiols with some aromatic disulphides. 2,2'-Dipyridyl disulphide as a convenient active-site titrant for papain even in the presence of other thiols.

1. The u.v.-spectral characteristics of 5,5'-dithiobis-(2-nitrobenzoic acid) (Nbs(2)), 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py), 4,4'-dipyridyl disulphide (4-Py-S-S-4-Py), 5-mercapto-2-nitrobenzoic acid (Nbs), 2-thiopyridone (Py-2-SH) and 4-thiopyridone (Py-4-SH) were determined over a wide range of pH and used to calculate their acid dissociation constants. 2. The reactions of l-cysteine, 2-mercaptoethanol and papain with the above-mentioned disulphides were investigated spectrophotometrically in the pH range 2.5-8.5. 3. Under the conditions of concentration used in this study the reactions of both low-molecular-weight thiols with all three disulphides resulted in the stoicheiometric release of the thiol or thione fragments Nbs, Py-2-SH and Py-4-SH at all pH values. The rates of these reactions are considerably faster at pH8 than at pH4, which suggests that the predominant reaction pathway in approximately neutral media is nucleophilic attack of the thiolate ion on the unprotonated disulphide. 4. The reaction of papain with Nbs(2) is markedly reversible in the acid region, and the pH-dependence of the equilibrium constant for this system in the pH range 5-8 at 25 degrees C and I=0.1 is described by: [Formula: see text] 5. Papain reacts with both 2-Py-S-S-2-Py and 4-Py-S-S-4-Py in the pH range 2.5-8.5 to provide release of the thione fragments, stoicheiometric with the thiol content of the enzyme. 6. Whereas the ratios of the second-order rate constant for the reaction at pH4 to that at pH8 for the cysteine-2-Py-S-S-2-Py reaction (k(pH4)/k(pH8)=0.015) and for the papain-4-Py-S-S-4-Py reaction (k(pH4)/k(pH8)=0.06) are less than 1, that for the papain-2-Py-S-S-2-Py reaction is greater than 1 (k(pH4)/k(pH8)=15). 7. This high reactivity of papain has been shown to involve reaction of the thiol group of cysteine-25, the enzyme's only cysteine residue, which is part of its catalytic site. 8. That this rapid and stoicheiometric reaction of the thiol group of native papain is not shown either by low-molecular-weight thiols or by the thiol group of papain after its active conformation has been destroyed by acid or heat denaturation, strongly commends 2-Py-S-S-2-Py as one of the most useful papain active-site titrants discovered to date. This reagent has been shown to allow accurate titration of papain active sites in the presence of up to 10-fold molar excess of l-cysteine and up to 100-fold molar excess of 2-mercaptoethanol.

Benzoates↗
PubMed1973

D-3-hydroxybutyrate dehydrogenase from Rhodopseudomonas spheroides. Kinetic mechanism from steady-state kinetics of the reaction catalysed by the enzyme in solution and covalently attached to diethylaminoethylcellulose.

1. The reversible NAD(+)-linked oxidation of d-3-hydroxybutyrate to acetoacetate in 0.1m-sodium pyrophosphate buffer, pH8.5, at 25.0 degrees C, catalysed by d-3-hydroxybutyrate dehydrogenase (d-3-hydroxybutyrate-NAD(+) oxidoreductase, EC 1.1.1.30), was studied by initial-velocity, dead-end inhibition and product-inhibition analysis. 2. The reactions were carried out on (a) the soluble enzyme from Rhodopseudomonas spheroides and (b) an insoluble derivative of this enzyme prepared by its covalent attachment to DEAE-cellulose by using 2-amino-4,6-dichloro-s-triazine as coupling agent. 3. The insolubilized enzyme preparation contained 5mg of protein/g wet wt. of total material, and when freshly prepared its specific activity was 1.2mumol/min per mg of protein, which is 67% of that of the soluble dialysed enzyme. 4. The reactions catalysed by both the enzyme in solution and the insolubilized enzyme were shown to follow sequential pathways in which the nicotinamide nucleotides bind obligatorily first to the enzyme. Evidence is presented for kinetically significant ternary complexes and that the rate-limiting step(s) of both catalyses probably involves isomerization of the enzyme-nicotinamide nucleotide complexes and/or dissociation of the nicotinamide nucleotides from the enzyme. Both catalyses therefore are probably best described as ordered Bi Bi mechanisms, possibly with multiple enzyme-nicotinamide nucleotide complexes. 5. The kinetic parameters and the calculable rate constants for the catalysis by the soluble enzyme are similar to the corresponding parameters and rate constants for the catalysis by the insolubilized enzyme.

Acetoacetates↗
PubMed1973

D-3-hydroxybutyrate dehydrogenase from Rhodopseudomonas spheroides. Kinetics of radioisotope redistribution at chemical equilibrium catalysed by the enzyme in solutions.

1. The reversible NAD(+)-linked oxidation of d-3-hydroxybutyrate to acetoacetate in 0.1m-sodium pyrophosphate buffer, pH8.5, at 25.0 degrees C, catalysed by d-3-hydroxybutyrate dehydrogenase (d-3-hydroxybutyrate-NAD(+) oxidoreductase, EC 1.1.1.30), was studied kinetically at chemical equilibrium by monitoring radioisotope redistribution with sodium dl-hydroxy[3-(14)C]butyrate and [4-(3)H]NAD(+)(labelled in the nicotinamide ring). 2. When all substrates are maintained at concentrations approaching saturation (approx. 3-50 times the K(m) values) the first-order rate constant for the enzyme-catalysed interconversion of NAD(+) and NADH is much smaller than that for the enzyme-catalysed interconversion of d-3-hydroxybutyrate and acetoacetate. 3. The rate of interconversion of NAD(+) and NADH increases initially with increasing concentrations of d-3-hydroxybutyrate and acetoacetate (ratio of concentrations maintained constant), passes through a maximum and approaches closely to zero at saturating concentrations of the latter substrates. 4. The rates of interconversion of NAD(+) and NADH and of d-3-hydroxybutyrate and acetoacetate increase with increasing concentration of NAD(+) (up to 66 times its K(m) value) and NADH (up to 180 times its K(m) value) (ratio of the concentrations of the nicotinamide nucleotides maintained constant). 5. These findings support the description of this catalysis as an ordered Bi Bi mechanism with no detectable alternative pathway, in which the interconversion of the central ternary complexes is not rate-limiting, and provide no evidence for the formation of dead-end complexes. 6. The solubility of 2,4-dinitrophenylhydrazine in HCl exhibits an acidity optimum, the maximum solubility at 25.0 degrees C (3.8mg/ml, 19mm) occurring at 2.29m-HCl; in solutions of this acidity acetone 2,4-dinitrophenylhydrazone is relatively insoluble (0.098mg/ml, 0.413mm).

Acetoacetates↗
PubMed1973

Covalent chromatography. Preparation of fully active papain from dried papaya latex.

1. A Sepharose-(glutathione-2-pyridyl disulphide) conjugate has been prepared. 2. Its use in a new type of chromatography, covalent chromatography by thiol-disulphide interchange, is described. 3. With this technique, papain containing 1 intact catalytic site [thiol with high reactivity towards 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py) at pH4] per mol of protein is readily prepared both from dried papaya latex and from commercial 2xcrystallized partially active papain. 4. The catalysis of the hydrolysis of alpha-N-benzoyl-l-arginine ethyl ester at pH6.0, 25.0 degrees C, I=0.3 by fully active papain thus prepared is characterized by K(m)=18.2+/-<0.1mm and k(cat.)=16.4+/-0.5s(-1).

Chromatography↗
PubMed1972

The reaction of papain with Ellman's reagent (5,5'-dithiobis- (2-nitrobenzoate) dianion).

1. The report by Robyt et al. (1971) that the 2-nitro-5-mercaptobenzoate dianion (Nbs(2-)) produced by reaction of papain with the 5,5'-dithiobis-(2-nitrobenzoate) dianion (Nbs(2) (2-); Ellman's reagent) cleaves the three disulphide bonds in papain is shown to be incorrect. 2. When partially active papain containing approx. 0.4 mol of thiol/mol of protein is incubated with excess of Nbs(2) (2-) at pH8, Nbs(2) (2-) reacts with the protein in an amount stoicheiometric with the cysteinyl thiol group of papain to produce Nbs(2-) in an amount stoicheiometric with the original papain cysteinyl thiol group, and the catalytically inactive mixed disulphide, papain-Nbs(-). 3. Papain catalyses the hydrolysis of Nbs(2) (2-) at pH10.5 probably by nucleophilic catalysis involving the enzyme's thiol group. 4. These results cast very serious doubts on the claim by Robyt et al. (1971) to have established a new general method for the determination of cystinyl disulphide residues in proteins.

Benzoates↗