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E M Crook

Publications and source records attributed to E M Crook.

At least 19 recordsLinked to original sources
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

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↗
PubMed1966

The preparation and properties of ficin chemically attached to carboxymethylcellulose.

1. Purified ficin has been coupled to four CM-celluloses by reaction with their acid azide derivatives. Insoluble products containing 1.8-4.7mg. of ficin/100mg. of product and retaining 8.0-12.0% of the free enzyme's esterase activity have been obtained. 2. The amount of bound ficin in these preparations is dependent on the degree of carboxymethyl substitution of the CM-cellulose to which the ficin is attached. 3. A shift of the alkaline limb of the pH-activity curve of ficin when chemically attached to CM-cellulose has been shown. 4. Only a small loss has been observed in the enzymic activity of these products when stored at 2 degrees for 4 months. They are more resistant than free enzyme to heat denaturation. 5. Columns of CM-cellulose-ficin have been packed. The degree of hydrolysis of perfused substrate has been measured for different flow rates through the column. 6. The properties of these derivatives have been discussed.

Chemical Phenomena↗
PubMed1966

The kinetics of carboxymethylcellulose--ficin in packed beds.

1. The kinetics of the hydrolysis of benzoylarginine ethyl ester in packed columns of CM-cellulose-70-ficin and CM-cellulose-90-ficin were studied. 2. The apparent Michaelis constant, K'(m), of these preparations was calculated and shown to be dependent on the flow rate at low rates of perfusion through the columns. 3. The values for k(3) of these preparations were calculated and shown to be nearly independent of flow rate. 4. A modified form of the integrated Michaelis rate equation was used to describe the action of these materials and its limitations are discussed. 5. The hydrolysis of solutions of casein by these columns was studied.

Arginine↗