scholarly journals A kinetic and fluorimetric investigation of papain modified at tryptophan -69 and -177 by N–bromosuccinimide

1974 ◽  
Vol 141 (2) ◽  
pp. 503-515 ◽  
Author(s):  
Gordon Lowe ◽  
Alan S. Whitworth
Keyword(s):  
Fluorescence Intensity ◽  
Fluorescence Spectra ◽  
Close Contact ◽  
Ph Dependence ◽  
Thiol Group ◽  
Structural Features ◽  
Enzymic Activity ◽  
Ph Profile ◽  
Hydrolysis Of ◽  
Nitrophenyl Ester

A systematic study of the modification of papain (its thiol group protected as a disulphide with mercaptoethanol) by N-bromosuccinimide, showed that 2 molar equiv. modified tryptophan-69 and 4 molar equiv. modified tryptophan -69 and -177. The Michaelis parameters for the catalysed hydrolysis of N-benzyloxycarbonylglycine p-nitrophenyl ester by these modified enzymes were determined. The enzymic activity of the modified enzymes was not seriously impaired, but modification of tryptophan-177 raised the apparent pKa of the acidic limb of the pH profile by more than 1 pH unit for both kcat. and kcat./Km. The fluorescence spectra (excitation at 288nm) of the modified enzymes showed that tryptophan -69 contributed about 8% to the fluorescence intensity, whereas tryptophan-177 contributed about 46% at neutral pH. However, the contribution of tryptophan -177 was quenched at low pH and its fluorescence intensity showed sigmoidal pH-dependence, with an apparent pKa of 4.2. Histidine -159, which is in close contact with tryptophan -177, is considered to be the residue responsible for the fluorescence quenching. When tryptophan -177 was modified, presumably generating a less hydrophobic micro-environment, the apparent pKa determined kinetically was raised to about 5.4. By comparing the Michaelis parameters of native papain, papain modified at tryptophan-69 and papain modified at tryptophan-69 and -177 with N–benzyloxycarbonylglycylglycine amide and N–benzyloxycarbonylglycyltryptophan amide, tryptophan-69 and tryptophan -177 were shown to be structural features of the S2 and S1′ subsites respectively.

scholarly journals Cathepsin B, the lysosomal thiol proteinase of calf liver

1969 ◽  
Vol 114 (4) ◽  
pp. 673-678 ◽  
Author(s):  
O. Snellman
Keyword(s):  
Cathepsin B ◽  
Ph Dependence ◽  
Gel Filtration ◽  
Chelating Agent ◽  
Thiol Group ◽  
Maximum Activity ◽  
Active Centre ◽  
Peptide Bonds ◽  
Hydrolysis Of ◽  
Thiol Proteinase

Cathepsin B from calf liver was obtained by a method involving preparation of a lysosomal–mitochondrial pellet and treatment of this pellet with acetone. The material was extracted with an acid buffer, pH4·0, and then precipitated from the extract with acetone. The precipitate was dissolved in phosphate buffer, pH7·4, and subjected to gel filtration on Sephadex G-200 and G-100. The cathepsin B emerged in a range of molecular weight much lower than 50000 as a well-defined component. The purity of this material was checked by electrophoresis. To obtain maximum activity the enzyme had to be activated with a chelating agent and a reducing agent (i.e. EDTA and cysteine). A number of different substrates were used. The enzyme was active for the hydrolysis of both peptide bonds and ester bonds and had approximately equal reactivity in the two cases. The pH-dependence of the hydrolysis was the same with both substrates. The binding of the substrates was half-maximal at pH4·5 and at pH6·8. A thiol group occurred in the active centre but this group ought to have a much higher pK than that found in this enzyme.

scholarly journals Subsite differences between the active centres of papaya peptidase A and papain as revealed by affinity chromatography. Purification of papaya peptidase A by ionic-strength-dependent affinity adsorption on an immobilized peptide inhibitor of papain

1984 ◽  
Vol 219 (3) ◽  
pp. 727-733 ◽  
Author(s):  
P Schack ◽  
N C Kaarsholm
Keyword(s):  
Ionic Strength ◽  
Thiol Group ◽  
Peptide Inhibitor ◽  
Affinity Column ◽  
Sh Group ◽  
The One ◽  
Hydrolysis Of ◽  
Nitrophenyl Ester ◽  
Thiol Proteinase ◽  
Immobilized Peptide

An affinity column consisting of the specific peptide inhibitor of papain, Gly-Gly (O-benzyl)Tyr-Arg, attached to Sepharose was found to bind the active thiol proteinase papaya peptidase A specifically, but only at an ionic strength significantly higher than the one at which papain is bound. When a mixture of active papaya peptidase A and its irreversibly oxidized contaminant was applied to the column, the active enzyme was bound whereas the inactive material was not. The bound enzyme was released by deionized water and found to contain 1 mol of SH group/mol of protein. The different conditions required for the binding of the two enzymes to the immobilized peptide was shown to reflect different ionic-strength-dependences of the affinity of the two enzymes for the peptide in solution. Whereas the affinity of papain for the inhibitor appears to be insensitive to ionic strength over the range studied, that of papaya peptidase A is ionic-strength-dependent and always lower than that of papain. A rate assay is devised for papaya peptidase A with N-benzyloxycarbonylglycine p-nitrophenyl ester as the substrate at pH 5.5. After calibration against an active-site titration the assay yields the thiol-group concentration without interference from inactive contaminants. For the papaya peptidase A-catalysed hydrolysis of N-benzyloxycarbonylglycine p-nitrophenyl ester at pH 5.5 kcat. was found to be 16.7s-1, which is about 3 times the value found for the same reaction catalysed by papain.

Fluorescence Reagents for Labelling of Biomolecules. Part I. Synthesis and Spectral Characterization of 2- and 4-Substituted 9-Isothiocyanatoacridines

1994 ◽  
Vol 59 (1) ◽  
pp. 203-212 ◽  
Author(s):  
Dana Mazagová ◽  
Danica Sabolová ◽  
Pavol Kristian ◽  
Ján Imrich ◽  
Marián Antalík ◽  
...  
Keyword(s):  
Chemical Shift ◽  
Carbon Atom ◽  
Fluorescence Intensity ◽  
Fluorescence Spectra ◽  
Chemical Shifts ◽  
H Nmr ◽  
Hammett Constants ◽  
Hydrolysis Of ◽  
C Nmr

9-Isothiocyanatoacridines VIII - XIV were prepared from the corresponding 9-chloroacridines I - VII. The IR, 1H NMR, 13C NMR and fluorescence spectra of the products are given. The 13C NMR chemical shifts of the C-9 ipso carbon atom exhibit a trend that is in accord with the Hammett constants of substituents bonded to the C-2 carbon. Effect of these substituents on the chemical shift of C-NCS was only small. The dependence of hydrolysis of isothiocyanates VIII - XIV on pH of the medium was studied. It was found that 9-isothiocyanatoacridines do not undergo hydrolysis at pH 7 - 10. The relative fluorescence intensities (F/F0) of compounds VIII - XIV at pH 7.4 have been determined in comparison with that of 9-aminoacridine. No direct dependence between the fluorescence intensity and the polar character of substituents has been found.

Transient-phase studies of a trypsin-catalyzed reaction

1970 ◽  
Vol 48 (12) ◽  
pp. 1793-1802 ◽  
Author(s):  
H. P. Kasserra ◽  
K. J. Laidler
Keyword(s):  
Steady State ◽  
Ph Dependence ◽  
Enzyme Concentration ◽  
Alcohol Concentration ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Steady State Kinetics ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Nitrophenyl Ester

The stopped-flow technique has been used to study the pre-steady-state kinetics of the hydrolysis of N-carbobenzoxy-L-alanine-p-nitrophenyl ester catalyzed by trypsin. By working under conditions such that the enzyme concentration is much greater than that of the substrate, it has been possible to measure [Formula: see text] the rate constant for the conversion of the enzyme-substrate complex into the acyl enzyme. The pH dependence of [Formula: see text] reveals a pKb′ value of 6.9 for the conversion of complex into acyl enzyme, in agreement with deductions from steady-state investigations. The pH dependence of [Formula: see text] (equal to k−1 + k2)/k1) has also been determined. The results provide direct evidence for the existence of an enzyme-substrate complex for this reaction.The work has been done in various mixtures of water and isopropyl alcohol. The logarithms of the rate constants [Formula: see text] and [Formula: see text] vary linearly with 1/D, showing a decrease with increasing alcohol concentration; [Formula: see text] increases with alcohol concentration. The solvent results suggest that addition of alcohol affects the hydrophobic bonding in the protein and leads to unfolding of the enzyme.

scholarly journals Chymopapain A. Purification and investigation by covalent chromatography and characterization by two-protonic-state reactivity-probe kinetics, steady-state kinetics and resonance Raman spectroscopy of some dithioacyl derivatives

1986 ◽  
Vol 233 (1) ◽  
pp. 119-129 ◽  
Author(s):  
B S Baines ◽  
K Brocklehurst ◽  
P R Carey ◽  
M Jarvis ◽  
E Salih ◽  
...  
Keyword(s):  
Close Contact ◽  
Cysteine Proteinase ◽  
Ph Dependence ◽  
Resonance Raman ◽  
Thiol Group ◽  
Cysteine Residue ◽  
Catalytic Site ◽  
Cysteine Proteinases ◽  
Ph Values ◽  
Covalent Chromatography

Chymopapain A was isolated from the dried latex of papaya (Carica papaya) by ion-exchange chromatography followed by covalent chromatography by thiol-disulphide interchange. The latter procedure was used to produce fully active enzyme containing one essential thiol group per molecule of protein, to establish that the chymopapain A molecule contains, in addition, one non-essential thiol group per molecule and to recalculate the literature value of epsilon 280 for the enzyme as 36 000 M−1 × cm −1. The Michaelis parameters for the hydrolysis of L-benzoylarginine p-nitroanilide and of benzyloxy-carbonyl-lysine nitrophenyl ester at 25 degrees C, and I 0.1 at several pH values catalysed by chymopapain A, papaya proteinase omega, papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) were determined. Towards these substrates chymopapain A has kcat./km values similar to those of actinidin and of papaya proteinase omega and significantly lower than those of papain or ficin. The environment of the catalytic site of chymopapain A is markedly different from those of other cysteine proteinases studied to date, as evidenced by the pH-dependence of the second-order rate constant (k) for the reaction of the catalytic-site thiol group with 2,2′-dipyridyl disulphide. The striking bell-shaped component that is a characteristic feature of the reactions of S-/ImH+ (thiolate/imidazolium) ion-pair components of many cysteine-proteinase catalytic sites with the 2,2′-dipyridyl disulphide univalent cation is not present in the pH-k profile for the chymopapain A reaction. The result is consistent with the presence of an additional positive charge in, or near, the catalytic site that repels the cationic form of the probe reagent. Resonance Raman spectra were collected at pH values 2.5, 6.0 and 8.0 for each of the following dithioacyl derivatives of chymopapain A: N-benzoylglycine-, N-(Beta-phenylpropionl)glycine- and N-methoxycarbonylphenylalanylglycine-. The main conclusion of the spectral study is that in each case the acyl group binds as a single population known as conformer B in which the glycinic N atom is in close contact with the thiol S atom of the catalytic-site cysteine residue, as is the case also for papain and other cysteine proteinases studied. Thus the abnormal catalytic-site environment of chymopapain A detected by the reactivity-probe studies, which may have consequences for the acylation step of the catalytic act, does not perturb the conformation of the bound acyl group at the acyl-enzyme-intermediate stage of catalysis.

Sign in / Sign up

Export Citation Format

Share Document