scholarly journals Identification of a cysteine residue at the active site of Escherichia coli isocitrate lyase

1989 ◽  
Vol 261 (2) ◽  
pp. 431-435 ◽  
Author(s):  
H G Nimmo ◽  
F Douglas ◽  
C Kleanthous ◽  
D G Campbell ◽  
C MacKintosh
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Isocitrate Lyase ◽  
Cysteine Residue ◽  
Order Process ◽  
Reactive Group ◽  
Site Isolation ◽  
First Order ◽  
Complete Inactivation ◽  
Ph Range

Escherichia coli isocitrate lyase was inactivated by iodacetate in a pseudo-first-order process. Complete inactivation was associated with the incorporation of only one carboxymethyl group per enzyme subunit. The substrate and products of the enzyme protected against inactivation, suggesting that the reactive group may be located at the active site. Isolation and sequencing of a carboxymethylated peptide showed that the modified residue was a cysteine, in the sequence Cys-Gly-His-Met-Gly-Gly-Lys. The reactivity of isocitrate lyase to iodoacetate declined with pH, following a titration curve for a group of pKa 7.1. The Km of the enzyme for isocritrate declined over the same pH range.

scholarly journals Mechanistic and active-site studies on d(–)-mandelate dehydrogenase from Rhodotorula graminis

1992 ◽  
Vol 281 (1) ◽  
pp. 211-218 ◽  
Author(s):  
D P Baker ◽  
C Kleanthous ◽  
J N Keen ◽  
E Weinhold ◽  
C A Fewson
Keyword(s):  
Active Site ◽  
Complete Sequence ◽  
Histidine Residue ◽  
Extended Version ◽  
Order Process ◽  
First Order ◽  
Tryptic Digest ◽  
Complete Inactivation ◽  
Hydrolysis Of ◽  
High Voltage Electrophoresis

D(–)-Mandelate dehydrogenase, the first enzyme of the mandelate pathway in the yeast Rhodotorula graminis, catalyses the NAD(+)-dependent oxidation of D(–)-mandelate to phenylglyoxylate. D(–)-2-(Bromoethanoyloxy)-2-phenylethanoic acid [‘D(–)-bromoacetylmandelic acid’], an analogue of the natural substrate, was synthesized as a probe for reactive and accessible nucleophilic groups within the active site of the enzyme. D(–)-Mandelate dehydrogenase was inactivated by D(–)-bromoacetylmandelate in a psuedo-first-order process. D(–)-Mandelate protected against inactivation, suggesting that the residue that reacts with the inhibitor is located at or near the active site. Complete inactivation of the enzyme resulted in the incorporation of approx. 1 mol of label/mol of enzyme subunit. D(–)-Mandelate dehydrogenase that had been inactivated with 14C-labelled D(–)-bromoacetylmandelate was digested with trypsin; there was substantial incorporation of 14C into two tryptic-digest peptides, and this was lowered in the presence of substrate. One of the tryptic peptides had the sequence Val-Xaa-Leu-Glu-Ile-Gly-Lys, with the residue at the second position being the site of radiolabel incorporation. The complete sequence of the second peptide was not determined, but it was probably an N-terminally extended version of the first peptide. High-voltage electrophoresis of the products of hydrolysis of modified protein showed that the major peak of radioactivity co-migrated with N tau-carboxymethylhistidine, indicating that a histidine residue at the active site of the enzyme is the most likely nucleophile with which D(–)-bromoacetylmandelate reacts. D(–)-Mandelate dehydrogenase was incubated with phenylglyoxylate and either (4S)-[4-3H]NADH or (4R)-[4-3H]NADH and then the resulting D(–)-mandelate and NAD+ were isolated. The enzyme transferred the pro-R-hydrogen atom from NADH during the reduction of phenylglyoxylate. The results are discussed with particular reference to the possibility that this enzyme evolved by the recruitment of a 2-hydroxy acid dehydrogenase from another metabolic pathway.

scholarly journals Identification of a cysteine residue as the binding site for the dipyrromethane cofactor at the active site of Escherichia coli porphobilinogen deaminase

FEBS Letters ◽  
1988 ◽  
Vol 235 (1-2) ◽  
pp. 189-193 ◽  
Author(s):  
Peter M. Jordan ◽  
Martin J. Warren ◽  
Howard J. Williams ◽  
Neal J. Stolowich ◽  
Charles A. Roessner ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Active Site ◽  
Cysteine Residue ◽  
Porphobilinogen Deaminase

scholarly journals Affinity labelling with a deaminatively generated carbonium ion. Kinetics and stoicheiometry of the alkylation of methionine-500 of the lacZβ-galactosidase of Escherichia coli by β-d-galactopyranosylmethyl-p-nitrophenyltriazene

1978 ◽  
Vol 175 (2) ◽  
pp. 525-538 ◽  
Author(s):  
M L Sinnott ◽  
P J Smith
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Sequence Data ◽  
Free Enzyme ◽  
Irreversible Inhibitor ◽  
Methionine Residue ◽  
Complete Inactivation ◽  
Carbonium Ion ◽  
Affinity Labelling ◽  
Field Desorption Mass Spectrometry

1. beta-D-Galactopyranosylmethyl-p-nitrophenyltriazene is an active-site-directed irreversible inhibitor of Mg2+-bound and Mg2+-free lacZ beta-galactosidase from Escherichia coli. 2. The Mg2+-enzyme binds the inhibitor more tightly but the complex then decomposes less rapidly than is the case with Mg2+-free enzyme. 3. Loss of enzyme activity is a linear function of the fraction of enzyme protomers to which are attached beta-D-galactopranosyl[14C]methyl residues: complete inactivation of fully active enzyme results in incorporation of 0.91 equivalent of carbohydrate label per enzyme protomer. 4. When the beta-galactopyranosylmethyl cation is generated in the active site of Mg2+-enzyme, it is captured essentially completely by the protein, but in the active site of Mg2+-free enzyme it is only captured with an efficiency of 25%. 5. Labelled enzyme was carboxymethylated and digested with trypsin; acidic hydrolysis of the isolated tryptic peptide, and field-desorption mass spectrometry of the isolated radioactive derivative, showed it to be 2,5-dioxo-3[2-(beta-D-galactopyranosylmethylthio)ethyl]-1,6-trimethylenepiperazine. 6. This is considered to have arisen from labelling of the sulphur atom of a methionine residue adjacent to a proline residue. 7. The complete amino acid sequence of the molecule [Fowler & Zabin (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1507-1510] enables the labelled methionine residue to be identified as either Met-421 or Met-500. 8. Sequence data [Fowler, Zabin, Sinnott & Smith (1978) J. Biol. Chem. in the press] show the site of attack to be Met-500.

scholarly journals Evidence for an arginine residue at the coenzyme-binding site of Escherichia coli isocitrate dehydrogenase

1989 ◽  
Vol 261 (1) ◽  
pp. 301-304 ◽  
Author(s):  
J S McKee ◽  
H G Nimmo
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Isocitrate Dehydrogenase ◽  
Arginine Residue ◽  
Order Process ◽  
First Order ◽  
Coenzyme Binding ◽  
Phosphorylated Form ◽  
Specific Reagent ◽  
Coenzyme Binding Site

The arginine-specific reagent phenylglyoxal inactivated the active, dephosphorylated, form of Escherichia coli isocitrate dehydrogenase rapidly in a pseudo-first-order process. Both NADP+ and NADPH protected the enzyme against inactivation. Phenylglyoxal appeared to react with one arginine residue per subunit, and the extent of the reaction was proportional to the extent of the inactivation. In contrast, the phosphorylated form of isocitrate dehydrogenase did not react detectably with phenylglyoxal. The data indicate that the coenzyme-binding site of isocitrate dehydrogenase contains a reactive arginine residue that is protected by phosphorylation, and are consistent with the hypothesis that phosphorylation of the enzyme occurs close to or at its active site.

scholarly journals Succinylation and inactivation of 3-hydroxy-3-methylglutaryl-CoA synthase by succinyl-CoA and its possible relevance to the control of ketogenesis

1985 ◽  
Vol 232 (1) ◽  
pp. 37-42 ◽  
Author(s):  
D M Lowe ◽  
P K Tubbs
Keyword(s):  
Active Site ◽  
Control Mechanism ◽  
Cysteine Residue ◽  
Time Dependent ◽  
Dependent Manner ◽  
Acetyl Coa ◽  
Active Site Cysteine ◽  
Complete Inactivation ◽  
Thioester Bond ◽  
Active Site Cysteine Residue

Succinyl-CoA (3-carboxypropionyl-CoA) inactivates ox liver mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) in a time-dependent manner, which is partially prevented by the presence of substrates of the enzyme. The inactivation is due to the enzyme catalysing its own succinylation. Complete inactivation corresponds to about 0.5 mol of succinyl group bound/mol of enzyme dimer. The succinyl-enzyme linkage appears to be a thioester bond and is probably formed with the active-site cysteine residue that is normally acetylated by acetyl-CoA. Succinyl-CoA binds to 3-hydroxy-3-methylglutaryl-CoA synthase with a binding constant of 340 microM and succinylation occurs with a rate constant of 0.57 min-1. Succinyl-enzyme breaks down with a half-life of about 40 min (k = 0.017 min-1) at 30 degrees C and pH 7 and is destabilized by the presence of acetyl-CoA and succinyl-CoA. A control mechanism is postulated in which flux through the 3-hydroxy-3-methylglutaryl-CoA cycle of ketogenesis is regulated according to the extent of succinylation of 3-hydroxy-3-methylglutaryl-CoA synthase.

The rate of exchange of propylenediaminetetraacetic acid with its cadmium complex

1966 ◽  
Vol 19 (12) ◽  
pp. 2213 ◽  
Author(s):  
B Bosnich ◽  
FP Dwyer ◽  
AM Sargeson
Keyword(s):  
Reaction Kinetics ◽  
Ethylenediaminetetraacetic Acid ◽  
Exchange Process ◽  
Optically Active ◽  
Second Order ◽  
Cadmium Complex ◽  
Order Process ◽  
First Order ◽  
Ph Range ◽  
Second Order Process

The polarimetric rates of exchange between optically active propylene- diaminetetraacetic acid (PDTA) and its cadmium complex, and that between ethylenediaminetetraacetic acid (EDTA) and the cadmium propylenediaminetetraacetato complex, have been measured in the pH range 4.75-7.00. An analysis of the reaction kinetics indicates that the exchange, in either case, occurs simultaneously by a first-order and second-order process, and it is concluded that both bimolecular and unimolecular pathways are operative in the exchange process.

scholarly journals Peroxide oxidation of indole to oxindole by chloroperoxidase catalysis

1979 ◽  
Vol 183 (2) ◽  
pp. 269-276 ◽  
Author(s):  
M D Corbett ◽  
B R Chipko
Keyword(s):  
Active Site ◽  
Maximal Activity ◽  
Major Product ◽  
Optimal Conditions ◽  
Peroxide Oxidation ◽  
Strong Inhibitor ◽  
Weak Inhibitor ◽  
Enzyme Active Site ◽  
First Order ◽  
Ph Range

In the presence of chloroperoxidase, indole was oxidized by H2O2 to give oxindole as the major product. Under most conditions oxindole was the only product formed, and under optimal conditions the conversion was quantitative. This reaction displayed maximal activity at pH 4.6, although appreciable activity was observed throughout the entire pH range investigated, namely pH 2.5-6.0. Enzyme saturation by indole could not be demonstrated, up to the limit of indole solubility in the buffer. The oxidation kinetics were first-order with respect to indole up to 8 mM, which was the highest concentration of indole that could be investigated. On the other hand, 2-methylindole was not affected by H2O2 and chloroperoxidase, but was a strong inhibitor of indole oxidation. The isomer 1-methylindole was a poor substrate for chloroperoxidase oxidation, and a weak inhibitor of indole oxidation. These results suggest the possibility that chloroperoxidase oxidation of the carbon atom adjacent to the nitrogen atom in part results from hydrogen-bonding of the substrate N-H group to the enzyme active site.

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