scholarly journals Multiple active conformers of mouse ornithine decarboxylase

1993 ◽  
Vol 293 (1) ◽  
pp. 289-295 ◽  
Author(s):  
S E Tsirka ◽  
C W Turck ◽  
P Coffino
Keyword(s):  
Conformational Change ◽  
Ornithine Decarboxylase ◽  
Enzymic Activity ◽  
Native Enzyme ◽  
Cysteine Residues ◽  
Natural Substrate ◽  
Guanidinium Chloride ◽  
Urea Treatment ◽  
Suicide Substrate ◽  
Kinetics Of

Purified recombinant mouse ornithine decarboxylase (ODC) was denatured with urea or with guanidinium chloride. Enzymic activity was efficiently recovered upon dilution of the denaturing agent. ODC renatured after urea treatment was further characterized. Kinetics of decarboxylation of the natural substrate ornithine or of the suicide substrate alpha-difluoromethylornithine (DFMO) were not significantly changed by denaturation/renaturation. Surprisingly, the renatured enzyme was not stably labelled with radioactive DFMO, in contrast with the native enzyme not subjected to denaturation. Native and renatured ODC did not differ in their c.d. spectra, but the former contained four reactive cysteine residues and the latter seven. These data indicate that a conformational change results from denaturation/renaturation that does not alter decarboxylation of substrates, but does change the accessibility or stability of the cysteine-360 residue modified by decarboxylated DFMO.

scholarly journals Conformational changes in the extracellular β-lactamase I from Bacillus cereus 569/H/9

1974 ◽  
Vol 143 (1) ◽  
pp. 137-141 ◽  
Author(s):  
Richard B. Davies ◽  
E. P. Abraham ◽  
D. G. Dalgleish
Keyword(s):  
Bacillus Cereus ◽  
Conformational Change ◽  
Conformational Changes ◽  
Thermal Denaturation ◽  
Complete Recovery ◽  
Enzymic Activity ◽  
Active Enzyme ◽  
Guanidinium Chloride ◽  
Subsequent Removal ◽  
Circular Dichroïsm

1. The thermal denaturation and precipitation of β-lactamase I from Bacillus cereus 569/H/9 at 60°C are reversible, a soluble and almost fully active enzyme being obtained after solution of the precipitate in 5m-guanidinium chloride or 8m-urea and subsequent removal of the denaturing agent. 2. Inactivation of β-lactamase I occurs rapidly between 50° and 55°C and is shown by circular-dichroism spectra to be accompanied by an extensive conformational change. 3. A change to a different conformation occurs in 6m-urea. This change is also reversible; refolding with almost complete recovery of enzymic activity occurs within 5min of dilution of the denaturing agent. 4. Inactivation of β-lactamase I at pH3.0 and 11.0 is also associated with conformational changes, since a proportion of the lost activity is recovered within 5min of adjustment of the pH to 7.0.

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.

Inactivation Kinetics of β-N-Acetyl-D-glucosaminidase from Green Crab (Scylla serrata) by Guanidinium Chloride

2012 ◽  
Vol 19 (11) ◽  
pp. 1177-1182
Author(s):  
Ji-Ping Zhang ◽  
Bo Leng ◽  
Qian-Sheng Huang ◽  
Ya-Wen Yan ◽  
Xuan Liu ◽  
...  
Keyword(s):  
Green Crab ◽  
Scylla Serrata ◽  
Inactivation Kinetics ◽  
Guanidinium Chloride ◽  
Kinetics Of

scholarly journals Reaction between sheep liver mitochondrial aldehyde dehydrogenase and various thiol-modifying reagents

1989 ◽  
Vol 261 (1) ◽  
pp. 281-284 ◽  
Author(s):  
K M Loomes ◽  
T M Kitson
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Related Compound ◽  
Physiological Responses ◽  
Enzymic Activity ◽  
Cysteine Residues ◽  
Mitochondrial Enzyme ◽  
Step Process ◽  
Cytoplasmic Enzyme ◽  
Sheep Liver

Sheep liver mitochondrial aldehyde dehydrogenase reacts with 2,2′-dithiodipyridine and 4,4′-dithiodipyridine in a two-step process: an initial rapid labelling reaction is followed by slow displacement of the thiopyridone moiety. With the 4,4′-isomer the first step results in an activated form of the enzyme, which then loses activity simultaneously with loss of the label (as has been shown to occur with the cytoplasmic enzyme). With 2,2′-dithiodipyridine, however, neither of the two steps of the reaction has any effect on the enzymic activity, showing that the mitochondrial enzyme possesses two cysteine residues that must be more accessible or reactive (to this reagent at least) than the postulated catalytically essential residue. The symmetrical reagent 5,5′-dithiobis-(1-methyltetrazole) activates mitochondrial aldehyde dehydrogenase approximately 4-fold, whereas the smaller related compound methyl l-methyltetrazol-5-yl disulphide is a potent inactivator. These results support the involvement of mixed methyl disulphides in causing unpleasant physiological responses to ethanol after the ingestion of certain antibiotics.

Degradation of ornithine decarboxylase: exposure of the C-terminal target by a polyamine-inducible inhibitory protein

1993 ◽  
Vol 13 (4) ◽  
pp. 2377-2383
Author(s):  
X Li ◽  
P Coffino
Keyword(s):  
Conformational Change ◽  
Ornithine Decarboxylase ◽  
Binding Protein ◽  
Active Form ◽  
Feedback Regulation ◽  
Degradation Process ◽  
Protein Inhibitor ◽  
Inhibitory Protein ◽  
C Terminus ◽  
N Terminus

Polyamine-mediated degradation of vertebrate ornithine decarboxylase (ODC) is associated with the production of antizyme, a reversible tightly binding protein inhibitor of ODC activity. The interaction of antizyme with a binding element near the N terminus of ODC is essential but not sufficient for regulation of the enzyme by polyamines (X. Li and P. Coffino, Mol. Cell. Biol. 12:3556-2562, 1992). We now show that a second element present at the C terminus is required for the degradation process. Antizyme caused a conformational change in ODC, which made the C terminus of ODC more accessible. Blocking the C terminus with antibody prevented degradation. Tethering the C terminus by creating a circularly permuted, enzymatically active form of ODC prevented antizyme-mediated degradation. These data elucidate a form of feedback regulation whereby excess polyamines induce destruction of ODC, the enzyme that initiates their biosynthesis.

scholarly journals Positive co-operative binding at two weak lysine-binding sites governs the Glu-plasminogen conformational change

1992 ◽  
Vol 285 (2) ◽  
pp. 419-425 ◽  
Author(s):  
U Christensen ◽  
L Mølgaard
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Conformational Change ◽  
Conformational Changes ◽  
Binding Sites ◽  
High Specificity ◽  
Stopped Flow ◽  
Protein Fluorescence ◽  
Lysine Residues ◽  
Kinetics Of

The kinetics of a series of Glu-plasminogen ligand-binding processes were investigated at pH 7.8 and 25 degrees C (in 0.1 M-NaCl). The ligands include compounds analogous to C-terminal lysine residues and to normal lysine residues. Changes of the Glu-plasminogen protein fluorescence were measured in a stopped-flow instrument as a function of time after rapid mixing of Glu-plasminogen and ligand at various concentrations. Large positive fluorescence changes (approximately 10%) accompany the ligand-induced conformational changes of Glu-plasminogen resulting from binding at weak lysine-binding sites. Detailed studies of the concentration-dependencies of the equilibrium signals and the rate constants of the process induced by various ligands showed the conformational change to involve two sites in a concerted positive co-operative process with three steps: (i) binding of a ligand at a very weak lysine-binding site that preferentially, but not exclusively, binds C-terminal-type lysine ligands, (ii) the rate-determining actual-conformational-change step and (iii) binding of one more lysine ligand at a second weak lysine-binding site that then binds the ligand more tightly. Further, totally independent initial small negative fluorescence changes (approximately 2-4%) corresponding to binding at the strong lysine-binding site of kringle 1 [Sottrup-Jensen, Claeys, Zajdel, Petersen & Magnusson (1978) Prog. Chem. Fibrinolysis Thrombolysis 3, 191-209] were observed for the C-terminal-type ligands. The finding that the conformational change in Glu-plasminogen involves two weak lysine-binding sites indicates that the effect cannot be assigned to any single kringle and that the problem of whether kringle 4 or kringle 5 is responsible for the process resolves itself. Probably kringle 4 and 5 are both participating. The involvement of two lysine binding-sites further makes the high specificity of Glu-plasminogen effectors more conceivable.

scholarly journals Ligand binding on to maize (Zea mays) malate synthase: a structural study

1994 ◽  
Vol 303 (2) ◽  
pp. 413-421 ◽  
Author(s):  
S Beeckmans ◽  
A S Khan ◽  
L Kanarek ◽  
E Van Driessche
Keyword(s):  
Zea Mays ◽  
Ligand Binding ◽  
Conformational Change ◽  
Limited Proteolysis ◽  
Enzymic Activity ◽  
Malate Synthase ◽  
Acetyl Coa ◽  
Original Activity ◽  
Kinetic Measurements ◽  
Michaelis Constants

A kinetic and ligand binding study on maize (Zea mays) malate synthase is presented. It is concluded from kinetic measurements that the enzyme proceeds through a ternary-complex mechanism. Michaelis constants (Km,glyoxylate and Km,acetyl-CoA) were determined to be 104 microM and 20 microM respectively. C.d. measurements in the near u.v.-region indicate that a conformational change is induced in the enzyme by its substrate, glyoxylate. From these studies we are able to calculate the affinity for the substrate (Kd,glyoxylate) as 100 microM. A number of inhibitors apparently trigger the same conformational change in the enzyme, i.e. pyruvate, glycollate and fluoroacetate. Another series of inhibitors bearing more bulky groups and/or an extra carboxylic acid also induce a conformational change, which is, however, clearly different from the former one. Limited proteolysis with trypsin results in cleavage of malate synthase into two fragments of respectively 45 and 19 kDa. Even when no more intact malate synthase chains are present, the final enzymic activity still amounts to 30% of the original activity. If trypsinolysis is performed in the presence of acetyl-CoA, the cleavage reaction is appreciably slowed down. The dissociation constant for acetyl-CoA (Kd,acetyl-CoA) was calculated to be 14.8 microM when the glyoxylate subsite is fully occupied by pyruvate and 950 microM (= 50 x Km) when the second subsite is empty. It is concluded that malate synthase follows a compulsory-order mechanism, glyoxylate being the first-binding substrate. Glyoxylate triggers a conformational change in the enzyme and, as a consequence, the correctly shaped binding site for acetyl-CoA is created. Demetallization of malate synthase has no effect on the c.d. spectrum in the near u.v.-region. Moreover, glyoxylate induces the same spectral change in the absence of Mg2+ as in its presence. Nevertheless, malate synthase shows no activity in the absence of the cation. We conclude that Mg2+ is essential for catalysis, rather than for the structure of the enzyme's catalytic site.

scholarly journals Rat liver cytosol contains NADPH- and GSH-dependent factors able to restore ornithine decarboxylase inactivated by removal of thiol reducing agents

1988 ◽  
Vol 250 (1) ◽  
pp. 53-58 ◽  
Author(s):  
F Flamigni ◽  
C Guarnieri ◽  
C M Caldarera
Keyword(s):  
Ornithine Decarboxylase ◽  
Rat Liver ◽  
Gel Filtration ◽  
Enzymic Activity ◽  
Effective Concentration ◽  
The Other ◽  
Limited Effect ◽  
Liver Cytosol ◽  
Rat Liver Cytosol ◽  
Dependent Mechanism

Removal of dithiothreitol (DTT) from partially purified ornithine decarboxylase (ODC) led to an almost complete inhibition of enzymic activity. The inactivation was reversed by addition of millimolar concentrations of DTT, whereas natural reductants such as NADPH or NADH were ineffective, and GSH had only a limited effect. Addition of rat liver cytosol to the incubation mixture resulted in a noticeable re-activation of ODC; however, dialysed cytosol had little effect unless NADPH or GSH was present. Fractionation of rat liver cytosol by gel filtration on Sephadex G-75 yielded two fractions involved in the NADPH- and GSH-dependent re-activation of ODC: one designated ‘A’, eluted near the void volume (Mr greater than or equal to 60,000), and the other designated ‘B’, eluted later (Mr approx. 12,000). The NADPH-dependent mechanism required both fractions A and B for maximal ODC re-activation; the most effective concentration of NADPH was 0.15 mM, although a significant effect was observed at a concentration more than 10-fold lower. The GSH-dependent mechanism involved the mediation of Fraction B only, and operated at millimolar concentrations of GSH. These results suggest the existence of reducing systems in the cytosol, which may play a role in maintaining, and potentially in regulating, ODC activity by modulation of its thiol status.

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