scholarly journals Bovine inositol monophosphatase. Modification, identification and mutagenesis of reactive cysteine residues

1992 ◽  
Vol 285 (2) ◽  
pp. 461-468 ◽  
Author(s):  
M R Knowles ◽  
N Gee ◽  
G McAllister ◽  
C I Ragan ◽  
P J Greasley ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Iodoacetic Acid ◽  
Native Enzyme ◽  
Steric Effects ◽  
Cysteine Residues ◽  
Native Protein ◽  
Protein Fluorescence ◽  
Inositol Monophosphatase ◽  
Nitrobenzoic Acid ◽  
Full Activity

1. Bovine inositol monophosphatase reacts with thiol reagents such as 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), N-ethylmaleimide (NEM) and iodoacetic acid (IAA). 2. Modification by NEM results in nearly total loss of enzyme activity, whereas modification by IAA causes a slight increase in activity. 3. The loss of activity caused by NEM can be prevented by the inclusion of Ins1P, or better Ins1P and LiCl in the reaction mixture. 4. Two equivalents of p-nitrothiobenzoate (NTB2-) are released from the native enzyme on reaction with DTNB, and six equivalents of NTB2- are released from the SDS-denatured enzyme, suggesting that none of the six cysteine residues per molecule of enzyme is involved in intra- or inter-molecular disulphide bridges. 5. Both NEM and IAA react with two cysteine residues (residues 141 and 184 in the sequence) in a mutually exclusive manner. 6. NEM also reacts stoichiometrically with residue 218. 7. The NEM-induced loss of enzyme activity is accompanied by a 15% decrease in protein fluorescence. 8. A mutant of the enzyme which has an Ala-218 replacement for Cys-218 has full activity and is not sensitive to NEM, showing that the modification of this cysteine by NEM causes inhibition of the native protein by steric effects and that Cys-218 is not essential for activity.

The Number, Location, and Reactivity of the Cysteine Residues of Sturgeon Muscle Aldolase

1972 ◽  
Vol 50 (2) ◽  
pp. 111-119 ◽  
Author(s):  
P. J. Anderson
Keyword(s):  
Iodoacetic Acid ◽  
Sulfhydryl Groups ◽  
Cysteine Residues ◽  
Rabbit Muscle ◽  
Nitrobenzoic Acid ◽  
Catalytic Role ◽  
Aldolase Activity ◽  
Activity Loss

Sturgeon muscle aldolase contains six cysteine residues per subunit. These residues appear to occur in homologous positions to six of the eight cysteine residues of rabbit muscle aldolase. Three of the six residues can react with either iodoacetic acid or 5,5′-dithiobis-(2-nitrobenzoic acid) in the absence of denaturing agents. Reaction of three residues with iodoacetic acid inactivates the enzyme. The presence of substrate protects one of these residues and in this case no activity loss is observed. However, the results indicate that the effects on activity are due to the addition of the modifying group rather than to a loss of sulfhydryl groups. No residue corresponding to the previously demonstrated substrate-protected cysteine of rabbit aldolase was located in the sturgeon enzyme, which demonstrated that this residue was not essential to aldolase activity. It therefore appears unlikely that cysteine residues have a direct or an auxiliary catalytic role in aldolase activity.

scholarly journals Carboxymethylation of Thiol Groups in Ovalbumin: Implications for Proteins that Contain Both Thiol and Disulfide Groups

1982 ◽  
Vol 35 (2) ◽  
pp. 125 ◽  
Author(s):  
DM Webster ◽  
EOP Thompson
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Guanidine Hydrochloride ◽  
Sodium Dodecyl ◽  
Iodoacetic Acid ◽  
Cysteine Residues ◽  
Alkaline Ph ◽  
Thiol Groups ◽  
Nitrobenzoic Acid ◽  
Dodecyl Sulfate ◽  
Fold Excess

The cysteine residues of hen ovalbumin were S-carboxymethylated with non-radioactive iodoacetic acid under various conditions by altering the pH at which the protein was denatured in 8 M urea, by using different molar ratios of non-radioactive iodoacetic acid to cysteine and by varying the time at which carboxymethylation was commenced after denaturing conditions had been applied. Under the various conditions, the thiol groups were carboxymethylated to different extents, the residual thiol groups being measured by reaction with 5,5'-dithiobis(2-nitrobenzoic acid) in the presence of sodium dodecyl sulfate. When ovalbumin is carboxymethylated in alkaline urea, it unfolds slowly and the carboxymethylation is incomplete even with 150-fold excess iodoacetic acid. The known rapid thiol-disulfide exchange that occurs at alkaline pH values makes this method of carboxymethylation unsuitable as a preliminary step for blocking the native cysteine residues of ovalbumin before reduction and labelling the thiol groups formed by reduction of the disulfide bonds. Titration of the thiol groups of ovalbumin in 6 M guanidine hydrochloride or 1 % (w/v) sodium dodecyl sulfate at pH 8�2 with 5,5' -dithiobis(2-nitrobenzoic acid) is more rapid than in 8 M urea and these solvents would be preferable for studies of the disulfide-bonded sequences. Denaturation of ovalbumin in acidic 8 M urea is a very rapid process, and under mild acid conditions thiol-disulfide interchange is much slower. Subsequent carboxymethylation of the cysteine residues at alkaline pH with 150-fold excess iodoacetic acid results in complete carboxymethylation and the carboxymethylated ovalbumin can be reduced and labelled with radioactive iodoacetic acid with specific labelling of the half-cystine residues involved in the disulfide bond. The results are discussed in relation to the allocation of half-cystine residues in other protein systems that contain both thiol and disulfide groups.

scholarly journals The reactivity of thiol groups and the subunit structure of aldolase

1970 ◽  
Vol 117 (2) ◽  
pp. 291-298 ◽  
Author(s):  
P. J. Anderson ◽  
R. N. Perham
Keyword(s):  
Group Analysis ◽  
Thiol Group ◽  
Enzymic Activity ◽  
Native Enzyme ◽  
Cysteine Residues ◽  
Subunit Structure ◽  
Rabbit Muscle ◽  
Thiol Groups ◽  
Prolonged Incubation ◽  
Nitrobenzoic Acid

1. Seven unique carboxymethylcysteine-containing peptides have been isolated from tryptic digests of rabbit muscle aldolase carboxymethylated with iodo[2-14C]acetic acid in 8m-urea. These peptides have been characterized by amino acid and end-group analysis and their location within the cyanogen bromide cleavage fragments of the enzyme has been determined. 2. Reaction of native aldolase with 5,5′-dithiobis-(2-nitrobenzoic acid), iodoacetamide and N-ethylmaleimide showed that a total of three cysteine residues per subunit of mol.wt. 40000 were reactive towards these reagents, and that the modification of these residues was accompanied by loss in enzymic activity. Chemical analysis of the modified enzymes demonstrated that the same three thiol groups are involved in the reaction with all these reagents but that the observed reactivity of a given thiol group varies with the reagent used. 3. One reactive thiol group per subunit could be protected when the modification of the enzyme was carried out in the presence of substrate, fructose 1,6-diphosphate, under which conditions enzymic activity was retained. This thiol group has been identified chemically and is possibly at or near the active site. Limiting the exposure of the native enzyme to iodoacetamide also served to restrict alkylation to two thiol groups and left the enzymic activity unimpaired. The thiol group left unmodified is the same as that protected by substrate during more rigorous alkylation, although it is now more reactive towards 5,5′-dithiobis-(2-nitrobenzoic acid) than in the native enzyme. 4. Conversely, prolonged incubation of the enzyme with fructose 1,6-diphosphate, which was subsequently removed by dialysis, caused an irreversible fall in enzymic activity and in thiol group reactivity measured with 5,5′-dithiobis-(2-nitrobenzoic acid). 5. It is concluded that the aldolase tetramer contains at least 28 cysteine residues. Each subunit appears to be identical with respect to number, location and reactivity of thiol groups.

Influence of carcinogens and group-specific compounds on DNAse II

1969 ◽  
Vol 47 (10) ◽  
pp. 987-989 ◽  
Author(s):  
Marvin S. Melzer
Keyword(s):  
Enzyme Activity ◽  
Enzyme Level ◽  
Iodoacetic Acid ◽  
Dnase Ii ◽  
Histidine Residues ◽  
Carcinogenic Process

A number of cancer-causing and group-specific compounds were tested for their effects on the activity of DNAse II. The following were almost completely inhibitory: iodoacetic acid, N-bromosuccinimide (at an N-bromosuccinimide/enzyme level ≥ 24), and H2O2 (at an H2O2/enzyme level > 10 000). Either noninhibitory or less than 30% inhibitory were iodoacetamide and diisopropylfluorophosphate. Noninhibitory were such carcinogens as beta-butyrolactone, diepoxybutane, 3-hydroxyxanthine, and ascaridole. Also noninhibitory was malonaldehyde.From these results (and others in the literature), it was concluded that (1) the carcinogens tested (at least in their unmetabolized forms) might not act directly on DNAse II in the critical reaction of the carcinogenic process, and (2) tryptophan, methionine, and/or histidine residues play important roles in the enzyme activity.

Effects of antisera raised against native and denatured human α-glucosidase and β-hexosaminidases on native enzyme activity

1984 ◽  
Vol 140 (3) ◽  
pp. 239-246 ◽  
Author(s):  
Anne-Marie Besançon ◽  
Sophie Gautron ◽  
Livia Poenaru ◽  
Jean-Claude Dreyfus
Keyword(s):  
Enzyme Activity ◽  
Native Enzyme

scholarly journals Mammalian inositol monophosphatase: the identification of residues important for the binding of Mg2+ and Li+ ions using fluorescence spectroscopy and site-directed mutagenesis

1993 ◽  
Vol 296 (3) ◽  
pp. 811-815 ◽  
Author(s):  
M G Gore ◽  
P Greasley ◽  
G McAllister ◽  
C I Ragan
Keyword(s):  
Fluorescence Spectroscopy ◽  
Specific Activity ◽  
Fluorescence Emission ◽  
Site Directed Mutagenesis ◽  
Native Enzyme ◽  
Mutant Enzyme ◽  
Directed Mutagenesis ◽  
Inositol Monophosphatase ◽  
Fluorescence Emission Intensity ◽  
Ph Dependent

The fluorescence properties of residue Trp-219 in inositol monophosphatase are sensitive to the ionization of neighbouring groups. The pH-dependent changes in the fluorescence emission intensity and wavelength of maximum emission appear to arise as the result of two separate ionizations in the proximity of Trp-219, namely due to the ionization of His-217 and Cys-218. By studying the curve of fluorescence intensity against pH, given by the mutants Cys-218→Ala or His-217→Gln, the pK of His-217 was determined to be 7.54 and the pK of Cys-218 was estimated to be about 8.2. These mutants have altered kinetic parameters for catalytic Mg2+ ions and inhibitory Mg2+ and Li+ ions. The Cys-218→Ala mutant enzyme is not subject to inhibition by concentrations of Mg2+ ions up to 400 mM and has a specific activity of 156% of the maximum obtainable activity of the native enzyme. The His-217→Gln mutant enzyme shows reduced sensitivity to inhibition by Mg2+ and Li+ ions, and has a specific activity of 110% of that obtainable for the native enzyme.

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