scholarly journals Probing the active site of glyoxalase I from human erythrocytes by use of the strong reversible inhibitor S-p-bromobenzylglutathione and metal substitutions

1981 ◽  
Vol 197 (1) ◽  
pp. 67-75 ◽  
Author(s):  
Anne-Charlotte Aronsson ◽  
Siv Sellin ◽  
Gudrun Tibbelin ◽  
Bengt Mannervik
Keyword(s):  
Catalytic Activity ◽  
Linear Regression Analysis ◽  
Strong Support ◽  
Human Erythrocytes ◽  
Reversible Inhibitor ◽  
Tryptophan Residue ◽  
Glyoxalase I ◽  
Enzyme Molecule ◽  
Binding Parameters ◽  
Thiol Groups

Glyoxalase I from human erythrocytes was studied by use of the strong reversible competitive inhibitor S-p-bromobenzylglutathione. Replacements of cobalt, manganese and magnesium for the essential zinc in the enzyme were made by a new procedure involving 10% methanol as a stabilizer of the enzyme. The Km value for the adduct of methylglyoxal and glutathione was essentially unchanged by the metal substitutions, whereas the inhibition constant for S-p-bromobenzylglutathione increased from 0.08μm for the Zn-containing enzyme to 1.3, 1.7 and 2.4μm for Co-, Mn- and Mg-glyoxalase I respectively. Binding of the inhibitor to the enzyme caused quenching of the tryptophan fluorescence of the protein, from which the binding parameters could be determined by the use of non-linear regression analysis. The highest dissociation constant was obtained for apoenzyme (6.9μm). The identity of the corresponding kinetic and binding parameters of the native enzyme and the Zn2+-re-activated apoenzyme and the clear differences from the parameters of the other metal-substituted enzyme forms give strong support to the previous identification of zinc as the natural metal cofactor of glyoxalase I. Binding to apoenzyme was also shown by the use of S-p-bromobenzylglutathione as a ligand in affinity chromatography and as a protector in chemical modification experiments. The tryptophan-modifying reagent 2-hydroxy-5-nitrobenzyl bromide caused up to 85% inactivation of the enzyme. After blocking of the thiol groups (about 8 per enzyme molecule) 6.1 2-hydroxy-5-nitrobenzyl groups were incorporated. Inclusion of S-p-bromobenzylglutathione with the modifying reagent preserved the catalytic activity of the enzyme completely and decreased the number of modified residues to 4.4 per enzyme molecule. The findings indicate the presence of one tryptophan residue in the active centre of each of the two subunits of the enzyme. Thiol groups appear not to be essential for catalytic activity. The presence of at least two categories of tryptophan residues in the protein was also shown by quenching of the fluorescence by KI.

scholarly journals Chemical modification of cellulase from Aspergillus niger

1977 ◽  
Vol 167 (3) ◽  
pp. 549-556 ◽  
Author(s):  
P L Hurst ◽  
P A Sullivan ◽  
M G Shepherd
Keyword(s):  
Ph Dependence ◽  
Cellulase Activity ◽  
Tryptophan Residue ◽  
Carboxyl Group ◽  
Enzyme Molecule ◽  
Thiol Groups ◽  
Original Activity ◽  
Activity Inhibition ◽  
Tryptophan Residues ◽  
Benzyl Halides

N-Bromosuccinimide completely inactivated the cellulase, and titration experiments showed that oxidation of one tryptophan residue per cellulase molecule coincided with 100% inactivation. CM-cellulose protected the enzyme from inactivation by N-bromosuccinimide. The cellulase was inhibited by active benzyl halides, and reaction with 2-hydroxy-5-nitrobenzyl bromide resulted in the incorporation of 2.3 hydroxy-5-nitrobenzyl groups per enzyme molecule; one tryptophan residue was shown to be essential for activity. Diazocarbonyl compounds in the presence of Cu2+ ions inhibited the enzyme. The pH-dependence of inactivation was consistent with the reaction occurring with a protonated carboxyl group. Carbodi-imide inhibited the cellulase, and kinetic analysis indicated that there was an average of 1 mol of carbodi-imide binding to the cellulase during inactivation. Treatment of the cellulase with diethyl pyrocarbonate resulted in the modification of two out of the four histidine residues present in the cellulase. The modified enzyme retained 40% of its original activity. Inhibition of cellulase activity by the metal ions Ag+ and Hg2+ was ascribed to interaction with tryptophan residues, rather than with thiol groups.

scholarly journals Half-sites oxidation of bovine liver uridine diphosphate glucose dehydrogenase

1978 ◽  
Vol 173 (2) ◽  
pp. 701-704 ◽  
Author(s):  
J S Franzen ◽  
P Marchetti ◽  
R Ishman ◽  
J Ashcom
Keyword(s):  
Catalytic Activity ◽  
Glucose Dehydrogenase ◽  
Bovine Liver ◽  
Thiol Group ◽  
Catalytic Site ◽  
Uridine Diphosphate ◽  
Thiol Groups ◽  
Irreversible Denaturation ◽  
Diphosphate Glucose ◽  
Uridine Diphosphate Glucose Dehydrogenase

6,6-Dithiodinicotinate shows half-of-the-sites reactivity towards the six catalytic-site thiol groups of bovine liver UDP-glucose dehydrogenase. The reagent introduces three intrasubunit disulphide linkages between catalytic-site thiol groups and non-catalytic-site thiol groups and abrogates 60% of the catalytic activity of the hexameric enzyme; excess 2-mercaptoethanol rapidly restores full catalytic activity. These results show the half-of-the-sites behaviour of the enzyme with the reagent and the presence of a non-catalytic-site thiol group capable of forming a disulphide linkage with a catalytic-site thiol group on the same subunit without irreversible denaturation.

scholarly journals Assimilation of α-glutamyl-peptides by human erythrocytes. A possible means of glutamate supply for glutathione synthesis

1985 ◽  
Vol 227 (3) ◽  
pp. 833-842 ◽  
Author(s):  
G F King ◽  
P W Kuchel
Keyword(s):  
Spin Echo ◽  
Linear Regression Analysis ◽  
Cell Suspensions ◽  
Human Erythrocytes ◽  
Peptide Hydrolysis ◽  
Whole Cell ◽  
Glutathione Synthesis ◽  
Whole Cells ◽  
Transmembrane Flux ◽  
Spin Echo Sequence

Human erythrocytes are essentially impermeable to glutamate and yet there is a continual requirement for the amino acid for glutathione synthesis. In addition, the intracellular glutamate concentration is approximately five times that of plasma. We present evidence that glutamate enters the red cell as small peptides which are rapidly hydrolysed by cytoplasmic peptidase(s) and that with the estimated physiological levels of plasma glutamyl-peptides the rate of inward flux would be adequate to maintain the glutamate pool at its observed level. Experimentally, we used 1H spin-echo n.m.r. spectroscopy to follow peptide hydrolysis, since peptide spectra are different from those of the free amino acids and the spin-echo sequence enables the monitoring of reactions in concentrated lysates and whole cell suspensions. Thus, the system was studied under near-physiological conditions. Weighted non-linear regression analysis of progress curves using the integrated Michaelis-Menten equation was used to obtain estimates of Km and Vmax. for the hydrolysis of alpha-L-glutamyl-L-alanine and L-alanyl-alpha-L-glutamate in lysates and whole cell suspensions; the values for lysates were Km = 3.60 +/- 0.29 and 5.4 +/- 0.4 mmol/l and Vmax. = 120 +/- 4 and 46.7 +/- 1.7 mmol/h per 1 of packed cells respectively. In whole cell suspensions the rate of peptide hydrolysis was much slower and dominated by the transmembrane flux-rate. The estimates of the steady-state kinetic parameters for the transport were Kt = 2.35 +/- 0.41 and 11.2 +/- 1.0 mmol/l and Vmax. = 3.26 +/- 0.13 and 19.7 +/- 0.7 mmol/h per 1 of packed cells respectively for the previously mentioned peptides. Using the n.m.r. procedure we failed to detect any glutaminase activity in whole cells or lysates; thus, we exclude the possibility that glutamate gains entry to the cell as glutamine which is subsequently hydrolysed by glutaminase.

ANTIOXIDANTS AS INHIBITORS OF LINOLEATE OXIDATION CATALYZED BY PLANT LIPOXIDASE AND BY HEMOLYZATES OF HUMAN ERYTHROCYTES

1955 ◽  
Vol 33 (1) ◽  
pp. 773-779 ◽  
Author(s):  
H. Bruce Collier ◽  
Sheila C. McRae
Keyword(s):  
Fatty Acids ◽  
Catalytic Activity ◽  
Erythrocyte Membrane ◽  
Unsaturated Fatty Acids ◽  
Human Erythrocytes ◽  
Catalytic Action ◽  
Time Activity ◽  
Linoleate Oxidation

Hemolyzates of human erythrocytes catalyzed the oxidation of linoleate at pH 7 but not at pH 9. Hence the erythrocytes contained no lipoxidase and the catalytic action was probably due to hemoglobin. However, the time-activity curves for hemolyzates and for crystalline hemoglobin were not identical in shape. The oxidation of linoleate at pH 7 by plant lipoxidase was powerfully inhibited by phenothiazine and by phenylhydrazine. These compounds, and also α-tocopherol and α-naphthol, inhibited the catalytic activity of hemolyzates and of crystalline hemoglobin. It is probable that phenothiazine and phenylhydrazine act as antioxidants in these systems. Antioxidants in vivo may possibly play a role in protecting the unsaturated fatty acids of the erythrocyte membrane from oxidation catalyzed by hemoglobin.

scholarly journals Thiol-dependent metallo-endopeptidase characteristics of Pz-peptidase in rat and rabbit

1990 ◽  
Vol 267 (2) ◽  
pp. 531-533 ◽  
Author(s):  
U Tisljar ◽  
A J Barrett
Keyword(s):  
Catalytic Activity ◽  
Rabbit Muscle ◽  
Thiol Groups ◽  
Rat Testis ◽  
Free Thiol

Pz-peptidase was purified from rat testis and rabbit muscle. Zinc was detectable in the rat enzyme. The activity of the enzyme from both species was slowly but completely abolished by EDTA and restored by Zn2+. Free thiol groups were also important for the catalytic activity of rat Pz-peptidase, as previously reported for the rabbit enzyme. We conclude that in both species Pz-peptidase has the characteristics of a thiol-dependent metallo-endopeptidase.

Population genetics of glyoxalase I (E.C.4.4.1.5) in human erythrocytes

1977 ◽  
Vol 79 (1) ◽  
Author(s):  
Konrad Berg ◽  
Alexander Rodewald ◽  
Friedrich Schwarzfischer ◽  
Hans Wischerath
Keyword(s):  
Population Genetics ◽  
Human Erythrocytes ◽  
Glyoxalase I

scholarly journals Chemical modification of Escherichia coli succinyl-CoA synthetase with the adenine nucleotide analogue 5′-p-fluorosulphonylbenzoyladenosine

1983 ◽  
Vol 215 (3) ◽  
pp. 513-518 ◽  
Author(s):  
A R S Prasad ◽  
J Ybarra ◽  
J S Nishimura
Keyword(s):  
Escherichia Coli ◽  
Catalytic Activity ◽  
Adenine Nucleotide ◽  
Bond Formation ◽  
Disulphide Bond ◽  
Thiol Groups ◽  
Binding Region ◽  
Nucleotide Analogue ◽  
Disulphide Bond Formation ◽  
Succinyl Coa Synthetase

Escherichia coli succinyl-CoA synthetase (EC 6.2.1.5) was irreversibly inactivated on incubation with the adenine nucleotide analogue 5′-p-fluorosulphonylbenzoyladenosine (5′-FSBA). Optimal inactivation by 5′-FSBA took place in 40% (v/v) dimethylformamide. ATP and ADP protected the enzyme against inactivation by 5′-FSBA, whereas desulpho-CoA, an analogue of CoA, did not. Inactivation of succinyl-CoA synthetase by 5′-FSBA resulted in total loss of almost four thiol groups per alpha beta-dimer, of which two groups appeared to be essential for catalytic activity. 5′-FSBA at the first instance appeared to interact non-specifically with non-essential thiol groups, followed by a more specific reaction with essential thiol groups in the ATP(ADP)-binding region. Plots of the data according to the method of Tsou [(1962) Sci. Sin. 11, 1535-1558] revealed that, of the two slower-reacting thiol groups, only one was essential for catalytic activity. When succinyl-CoA synthetase that had been totally inactivated by 5′-FSBA was unfolded in acidic urea and then refolded in the presence of 100 mM-dithiothreitol, 85% of the activity, in comparison with the appropriate control, was restored. These data are interpreted to indicate that inactivation of succinyl-CoA synthetase by 5′-FSBA involves the formation of a disulphide bond between two cysteine residues. Disulphide bond formation likely proceeds via a thiosulphonate intermediate between 5′-p-sulphonylbenzoyladenosine and one of the reactive thiol groups of the enzyme.

scholarly journals Probing the catalytic mechanism of Escherichia coli amine oxidase using mutational variants and a reversible inhibitor as a substrate analogue

2002 ◽  
Vol 365 (3) ◽  
pp. 809-816 ◽  
Author(s):  
Colin G. SAYSELL ◽  
Winston S. TAMBYRAJAH ◽  
Jeremy M. MURRAY ◽  
Carrie M. WILMOT ◽  
Simon E.V. PHILLIPS ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Amine Oxidase ◽  
Reaction Pathway ◽  
Strong Support ◽  
Reversible Inhibitor ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Binding Data ◽  
Copper Amine

Copper amine oxidases are homodimeric enzymes containing one Cu2+ ion and one 2,4,5-trihydroxyphenylalanine quinone (TPQ) per monomer. Previous studies with the copper amine oxidase from Escherichia coli (ECAO) have elucidated the structure of the active site and established the importance in catalysis of an active-site base, Asp-383. To explore the early interactions of substrate with enzyme, we have used tranylcypromine (TCP), a fully reversible competitive inhibitor, with wild-type ECAO and with the active-site base variants D383E and D383N. The formation of an adduct, analogous to the substrate Schiff base, between TCP and the TPQ cofactor in the active site of wild-type ECAO and in the D383E and D383N variants has been investigated over the pH range 5.5–9.4. For the wild-type enzyme, the plot of the binding constant for adduct formation (Kb) against pH is bell-shaped, indicating two pKas of 5.8 and ∼8, consistent with the preferred reaction partners being the unprotonated active-site base and the protonated TCP. For the D383N variant, the reaction pathway involving unprotonated base and protonated TCP cannot occur, and binding must follow a less favoured pathway with unprotonated TCP as reactant. Surprisingly, for the D383E variant, the Kb versus pH behaviour is qualitatively similar to that of D383N, supporting a reaction pathway involving unprotonated TCP. The TCP binding data are consistent with substrate binding data for the wild type and the D383E variant using steady-state kinetics. The results provide strong support for a protonated amine being the preferred substrate for the wild-type enzyme, and emphasize the importance of the active-site base, Asp-383, in the primary binding event.

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