scholarly journals Modification of lactate oxidase with diethyl pyrocarbonate. Evidence for an active-site histidine residue

1977 ◽  
Vol 165 (2) ◽  
pp. 385-393 ◽  
Author(s):  
Choong Yee Soon ◽  
Maxwell G. Shepherd ◽  
Patrick A. Sullivan
Keyword(s):  
Enzyme Activity ◽  
Chemical Reduction ◽  
Enzyme Inactivation ◽  
Subunit Structure ◽  
Histidine Residue ◽  
Lactate Oxidase ◽  
Histidine Residues ◽  
Diethyl Pyrocarbonate ◽  
Competitive Inhibitors ◽  
Active Site Histidine

1. Diethyl pyrocarbonate inactivated l-lactate oxidase from Mycobacterium smegmatis. 2. Two histidine residues underwent ethoxycarbonylation when the enzyme was treated with sufficient reagent to abolish more than 90% of the enzyme activity, but analyses of the inactivation showed that the modification of one histidine residue was sufficient to cause the loss of enzyme activity. The rates of enzyme inactivation and histidine modification were the same. 3. Substrate and competitive inhibitors decreased the maximum extent of inactivation to a 50% loss of enzyme activity and modification was decreased from 1.9 to 0.75–1.2 histidine residues modified/molecule of FMN. 4. Treatment of the enzyme with diethyl [14C]pyrocarbonate (labelled in the carbonyl groups) confirmed that only histidine residues were modified under the conditions used and that deacylation of the ethoxycarbonylhistidine residues by hydroxylamine was concomitant with the removal of the14C label and the re-activation of the enzyme. 5. No evidence was found for modification of tryptophan, tyrosine or cysteine residues, and no difference was detected between the conformation and subunit structure of the modified and native enzyme. 6. Modification of the enzyme with diethyl pyrocarbonate did not alter the following properties: the binding of competitive inhibitors, bisulphite and substrate or the chemical reduction of the flavin group to the semiquinone or fully reduced states. The normal reduction of the flavin by lactate was, however, abolished.

scholarly journals Modification of uridine phosphorylase from Escherichia coli by diethyl pyrocarbonate. Evidence for a histidine residue in the active site of the enzyme

1990 ◽  
Vol 270 (2) ◽  
pp. 319-323 ◽  
Author(s):  
A K Drabikowska ◽  
G Woźniak
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Active Site ◽  
Order Rate Constant ◽  
Enzymic Activity ◽  
Histidine Residue ◽  
Uridine Phosphorylase ◽  
High Concentration ◽  
Histidine Residues ◽  
Diethyl Pyrocarbonate

Uridine phosphorylase from Escherichia coli is inactivated by diethyl pyrocarbonate at pH 7.1 and 10 degrees C with a second-order rate constant of 840 M-1.min-1. The rate of inactivation increases with pH, suggesting participation of an amino acid residue with pK 6.6. Hydroxylamine added to the inactivated enzyme restores the activity. Three histidine residues per enzyme subunit are modified by diethyl pyrocarbonate. Kinetic and statistical analyses of the residual enzymic activity, as well as the number of modified histidine residues, indicate that, among the three modifiable residues, only one is essential for enzyme activity. The reactivity of this histidine residue exceeded 10-fold the reactivity of the other two residues. Uridine, though at high concentration, protects the enzyme against inactivation and the very reactive histidine residue against modification. Thus it may be concluded that uridine phosphorylase contains only one histidine residue in each of its six subunits that is essential for enzyme activity.

scholarly journals Inactivation of the endogenous argininosuccinate lyase activity of duck δ-crystallin by modification of an essential histidine residue with diethyl pyrocarbonate

1993 ◽  
Vol 293 (2) ◽  
pp. 537-544 ◽  
Author(s):  
H J Lee ◽  
S H Chiou ◽  
G G Chang
Keyword(s):  
Enzyme Activity ◽  
Rate Constant ◽  
Order Rate Constant ◽  
Histidine Residue ◽  
Argininosuccinate Lyase ◽  
First Order ◽  
Diethyl Pyrocarbonate ◽  
Order Rate ◽  
Lyase Activity ◽  
Pseudo First Order

The argininosuccinate lyase activity of duck delta-crystallin was inactivated by diethyl pyrocarbonate at 0 degrees C and pH 7.5. The inactivation followed pseudo-first-order kinetics after appropriate correction for the decomposition of the reagent during the modification period. The plot of the observed pseudo-first-order rate constant versus diethyl pyrocarbonate concentration in the range of 0.17-1.7 mM was linear and went through the origin with a second-order rate constant of 1.45 +/- 0.1 M-1.s-1. The double-logarithmic plot was also linear, with slope of 1.13, which suggested a 1:1 stoichiometry for the reaction between diethyl pyrocarbonate and delta-crystallin. L-Arginine, L-norvaline or L-citrulline protected the argininosuccinate lyase activity of delta-crystallin from diethyl pyrocarbonate inactivation. The dissociation constants for the delta-crystallin-L-arginine and delta-crystallin-L-citrulline binary complexes, determined by the protection experiments, were 4.2 +/- 0.2 and 0.12 +/- 0.04 mM respectively. Fumarate alone had no protective effect. However, fumarate plus L-arginine gave synergistic protection with a ligand binding interacting factor of 0.12 +/- 0.02. The double-protection data conformed to a random Uni Bi kinetic mechanism. Fluorescence-quenching studies indicated that the modified delta-crystallin had minimum, if any, conformational changes as compared with the native delta-crystallin. Inactivation of the enzyme activity was accompanied by an increasing absorbance at 240 nm of the protein. The absorption near 280 nm did not change. Treatment of the modified protein with hydroxylamine regenerated the enzyme activity to the original level. These results strongly indicated the modification of an essential histidine residue. Calculation from the 240 nm absorption changes indicated that only one histidine residue per subunit was modified by the reagent. This super-active histidine residue has a pKa value of approximately 6.8 and acts as a general acid-base catalyst in the enzyme reaction mechanism. Our experimental data are compatible with an E1cB mechanism [Raushel (1984) Arch. Biochem. Biophys. 232, 520-525] for the argininosuccinate lyase with the essential histidine residue close to the arginine-binding domain of delta-crystallin. L-Citrulline, after binding to this domain, might form an extra hydrogen bond with the essential histidine residue.

scholarly journals The reaction of a histidine residue in glutamate dehydrogenase with diethyl pyrocarbonate

1973 ◽  
Vol 133 (1) ◽  
pp. 183-187 ◽  
Author(s):  
Robert B. Wallis ◽  
J. John Holbrook
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Steady State ◽  
Glutamate Dehydrogenase ◽  
Specific Activity ◽  
Histidine Residue ◽  
Apparent Change ◽  
Diethyl Pyrocarbonate ◽  
Polypeptide Chains ◽  
Rate Limiting

1. One mol of diethyl pyrocarbonate will react with one mol of glutamate dehydrogenase polypeptide chains to form one mol of N1-carbethoxyhistidine. Reaction is prevented by NADH. 2. The 1:1 complex has an increased specific activity (1.4–2.0-fold). 3. The reason for the activation is discussed. The results are not consistent with NADH dissociation from the enzyme–glutamate–NADH complex being rate-limiting in the steady state measured. 4. The effects of modification on the properties of the enzyme were investigated. The effects of GTP and NAD+ on the enzyme activity are unaltered by activation. NADH binding is unaltered and there is no apparent change in the molecular weight. However, the activated enzyme can still be further activated by ADP. Ks for ADP is decreased fivefold.

Enthalpy and enzyme activity of modified histidine residues of adenosine deaminase and diethyl pyrocarbonate complexes

2000 ◽  
Vol 27 (1) ◽  
pp. 29-33 ◽  
Author(s):  
G. Ataie ◽  
A.A. Moosavi-Movahedi ◽  
A.A. Saboury ◽  
G.H. Hakimelahi ◽  
J.Ru. Hwu ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Adenosine Deaminase ◽  
Histidine Residues ◽  
Diethyl Pyrocarbonate

scholarly journals The histidine residues of phospholipase C from Bacillus cereus

1977 ◽  
Vol 167 (2) ◽  
pp. 399-404 ◽  
Author(s):  
C Little
Keyword(s):  
Catalytic Activity ◽  
Bacillus Cereus ◽  
Phospholipase C ◽  
Maximum Velocity ◽  
Native Enzyme ◽  
Histidine Residue ◽  
Histidine Residues ◽  
Residue Removal ◽  
Diethyl Pyrocarbonate ◽  
Km Value

The inactivation of phospholipase C from Bacillus cereus at pH6 by diethyl pyrocarbonate parallelled the N-ethoxyformylation of a single histidine residue in the enzyme. The inactivation arose from a decrease in the maximum velocity of the enzymic reaction with no effect on the Km value. The inactivation did not apparently alter the ability of the enzyme to bind to a substrate-based affinity gel. The native enzyme contained only one reactive histidine residue. Removal of the two zinc atoms from the enzyme increased the number of reactive histidine residues to five, whereas in the totally denatured enzyme nearly eight such residues were available for reaction with diethyl pyrocarbonate. The enzyme thus appears to contain one histidine residue that is essential for catalytic activity and four that may be involved in co-ordinating the zinc atoms in the structure.

scholarly journals Evidence for an essential histidine residue in d-xylose isomerases

1988 ◽  
Vol 250 (1) ◽  
pp. 153-160 ◽  
Author(s):  
W Vangrysperre ◽  
M Callens ◽  
H Kersters-Hilderson ◽  
C K De Bruyne
Keyword(s):  
Xylose Isomerase ◽  
Lactobacillus Brevis ◽  
Histidine Residue ◽  
Difference Spectra ◽  
Streptomyces Sp ◽  
Histidine Residues ◽  
Tyrosine Residues ◽  
Diethyl Pyrocarbonate ◽  
The Difference ◽  
Streptomyces Violaceoruber

Diethyl pyrocarbonate inactivated D-xylose isomerases from Streptomyces violaceoruber, Streptomyces sp., Lactobacillus xylosus and Lactobacillus brevis with second-order rate constants of 422, 417, 99 and 92 M-1.min-1 respectively (at pH 6.0 and 25 degrees C). Activity was completely restored by the addition of neutral hydroxylamine, and total protection was afforded by the substrate analogue xylitol in the presence of either Mg2+ or Mn2+ according to the genus studied. The difference spectra of the modified enzymes revealed an absorption maximum at 237-242 nm, characteristic for N-ethoxycarbonylhistidine. In addition, the spectrum of ethoxycarbonylated D-xylose isomerase from L. xylosus showed absorption minima at both 280 and 230 nm, indicative for modification of tyrosine residues. Nitration with tetranitromethane followed by diethyl pyrocarbonate treatment eliminated the possibility that modification of tyrosine residues was responsible for inactivation, and resulted in modification of one non-essential tyrosine residue and six histidine residues. Inactivation of the other D-xylose isomerases with diethyl pyrocarbonate required the modification of one (L. brevis), two (Streptomyces sp.) and four (S. violaceoruber) histidine residues per monomer. Spectral analysis and maintenance of total enzyme activities further indicated that either xylitol Mg2+ (streptomycetes) or xylitol Mn2+ (lactobacilli) prevented the modification of one crucial histidine residue. The overall results thus provide evidence that a single active-site histidine residue is involved in the catalytic reaction mechanism of D-xylose isomerases.

scholarly journals Histidine residues and the enzyme activity of pig heart supernatant malate dehydrogenase

1974 ◽  
Vol 139 (3) ◽  
pp. 797-800 ◽  
Author(s):  
J. John Holbrook ◽  
A. Lodola ◽  
Nicholas P. Illsley
Keyword(s):  
Enzyme Activity ◽  
Binding Site ◽  
Malate Dehydrogenase ◽  
Ternary Complex ◽  
Histidine Residues ◽  
Diethyl Pyrocarbonate

1. Supernatant pig heart malate dehydrogenase is completely inhibited by reaction with diethyl pyrocarbonate at pH6.5, when 0.58±0.1 residue of ethoxycarbonylhistidine is formed per NADH-binding site. 2. Oxaloacetate and hydroxymalonate protect the enzyme from inhibition in the absence of coenzyme. 3. Limited ethoxycarbonylation does not alter the binding of NADH to the enzyme but prevents the enzyme–NADH complex from interacting with hydroxymalonate in a ternary complex.

scholarly journals Malate dehydrogenase of the cytosol. Ionizations of the enzyme-reduced-coenzyme complex and a comparison with lactate dehydrogenase

1978 ◽  
Vol 173 (2) ◽  
pp. 597-605 ◽  
Author(s):  
A Lodola ◽  
D M Parker ◽  
R Jeck ◽  
J J Holbrook
Keyword(s):  
Lactate Dehydrogenase ◽  
Malate Dehydrogenase ◽  
Active Site ◽  
Histidine Residue ◽  
Histidine Residues ◽  
Coenzyme Binding ◽  
Active Site Histidine

1. The pH-dependencies of the binding of NADH and reduced nicotinamide–benzimidazole dinucleotide to pig heart cytoplasmic malate dehydrogenase and lactate dehydrogenase are reported. 2. Two ionizing groups were observed in the binding of both reduced coenzymes to lactate dehydrogenase. One group, with pKa in the range 6.3–6.7, is the active-site histidine residue and its deprotonation weakens binding of reduced coenzyme 3-fold. Binding of both coenzymes is decreased to zero when a second group, of pKa 8.9, deprotonates. This group is not cysteine-165.3. Only one ionization is required to characterize the binding of the two reduced coenzymes to malate dehydrogenase. The group involved appears to be the active-site histidine residue, since its ethoxycarbonylation inhibits the enzyme and abolishes binding of reduced coenzyme. Binding of either reduced coenzyme increases the pKa of the group from 6.4 to 7.4, and deprotonation of the group is accompanied by a 10-fold weakening of coenzyme binding. 4. Two reactive histidine residues were detected per malate dehydrogenase dimer. 5. A mechanism which emphasizes the homology between the two enzymes is presented.

scholarly journals Ionic properties of an essential histidine residue in pig heart lactate dehydrogenase

1973 ◽  
Vol 131 (4) ◽  
pp. 729-738 ◽  
Author(s):  
J. John Holbrook ◽  
V. Ann Ingram
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Lactate Dehydrogenase ◽  
Side Chains ◽  
Histidine Residue ◽  
Acylating Agent ◽  
Diethyl Pyrocarbonate ◽  
Substrate Analogues ◽  
Amino Acid Side Chains

1. Pig heart lactate dehydrogenase is inhibited by addition of one equivalent of diethyl pyrocarbonate. The inhibition is due to the acylation of a unique histidine residue which is 10-fold more reactive than free histidine. No other amino acid side chains are modified. 2. The carbethoxyhistidine residue slowly decomposes and the enzyme activity reappears. 3. The essential histidine residue is only slightly protected by the presence of NADH but is completely protected when substrate and substrate analogues bind to the enzyme–NADH complex. The protection is interpreted in terms of a model in which substrates can only bind to the enzyme in which the histidine residue is protonated and is thus not available for reaction with the acylating agent. 4. The apparent pKa of the histidine residue in the apoenzyme is 6.8±0.2. In the enzyme–NADH complex it is 6.7±0.2. 5. Acylated enzyme binds NADH with unchanged affinity. The enzyme is inhibited because substrates and substrate analogues cannot bind at the acylated histidine residue in the enzyme–NADH complex.

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