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.

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 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 Conformational changes on substrate binding revealed by structures of Methylobacterium extorquens malate dehydrogenase

2018 ◽  
Vol 74 (10) ◽  
pp. 610-616 ◽  
Author(s):  
Javier M. González ◽  
Ricardo Marti-Arbona ◽  
Julian C.-H. Chen ◽  
Brian Broom-Peltz ◽  
Clifford J. Unkefer
Keyword(s):  
Binding Site ◽  
Malate Dehydrogenase ◽  
Conformational Changes ◽  
Ternary Complex ◽  
Substrate Binding ◽  
Pyridine Nucleotide ◽  
Binary Complex ◽  
Methylobacterium Extorquens ◽  
Catalytic Complex ◽  
Α Helix

Three high-resolution X-ray crystal structures of malate dehydrogenase (MDH; EC 1.1.1.37) from the methylotroph Methylobacterium extorquens AM1 are presented. By comparing the structures of apo MDH, a binary complex of MDH and NAD+, and a ternary complex of MDH and oxaloacetate with ADP-ribose occupying the pyridine nucleotide-binding site, conformational changes associated with the formation of the catalytic complex were characterized. While the substrate-binding site is accessible in the enzyme resting state or NAD+-bound forms, the substrate-bound form exhibits a closed conformation. This conformational change involves the transition of an α-helix to a 310-helix, which causes the adjacent loop to close the active site following coenzyme and substrate binding. In the ternary complex, His284 forms a hydrogen bond to the C2 carbonyl of oxaloacetate, placing it in a position to donate a proton in the formation of (2S)-malate.

scholarly journals Evidence for a histidine and a cysteine residue in the substrate-binding site of yeast alcohol dehydrogenase

1975 ◽  
Vol 145 (3) ◽  
pp. 581-590 ◽  
Author(s):  
V Leskovac ◽  
D Pavkov-Peričin
Keyword(s):  
Alcohol Dehydrogenase ◽  
Binding Site ◽  
Ternary Complex ◽  
Substrate Binding ◽  
Cysteine Residue ◽  
Histidine Residue ◽  
Thiol Groups ◽  
Substrate Binding Site ◽  
Yeast Alcohol Dehydrogenase ◽  
Diethyl Pyrocarbonate

1. Yeast alcohol dehydrogenase (EC 1.1.1.1) is inhibited by stoicheiometric concentrations of diethyl pyrocarbonate. The inhibition is due to the acylation of a single histidine residue/monomer (mol.wt. 36000). 2. Alcohol dehydrogenase is also inhibited by stoicheiometric amounts of 5,5′-dithiobis-(2-nitrobenzoate), owing to the modification of a single cysteine residue/monomer. 3. Native alcohol dehydrogenase binds two molecules of reduced coenzyme/molecule of enzyme (mol.wt. 144000). 4. Modification of a single histidine residue/monomer by treatment with diethyl pyrocarbonate prevents the binding of acetamide in the ternary complex, enzyme-NADH-acetamede, but does not prevent the binding of NADH to the enzyme. 5. Modification of a single cysteine residue/monomer does not prevent the binding of acetamide to the ternary complex. After the modification of two thiol groups/monomer by treatment with 5,5′-dithiobis-(2-nitrobenzoate), the capacity of enzyme to bind coenzyme in the ternary complex was virtually abolished. 6. From the results presented in this paper we conclude that at least one histidine and one cysteine residue are closely associated in the substrate-binding site of alcohol dehydrogenase.

scholarly journals Amino acids important in enzyme activity and dimer stability for Drosophila alcohol dehydrogenase

1995 ◽  
Vol 308 (2) ◽  
pp. 419-423 ◽  
Author(s):  
S W Chenevert ◽  
N G Fossett ◽  
S H Chang ◽  
I Tsigelny ◽  
M E Baker ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Alcohol Dehydrogenase ◽  
Binding Site ◽  
Aspartic Acid ◽  
Three Dimensional ◽  
Side Chain ◽  
Wild Type ◽  
Dimensional Model ◽  
Three Dimensional Model ◽  
Substrate Binding Site

We have determined the nucleotide sequences of eight ethyl methanesulphonate-induced mutants in Drosophila alcohol dehydrogenase (ADH), of which six were previously characterized by Hollocher and Place [(1988) Genetics 116, 253-263 and 265-274]. Four of these ADH mutants contain a single amino acid change: glycine-17 to arginine, glycine-93 to glutamic acid, alanine-159 to threonine, and glycine-184 to aspartic acid. Although these mutants are inactive, three mutants (Gly17Arg, Gly93Glu and Gly184Asp) form stable homodimers, as well as heterodimers with wild-type ADH, in which the wild-type ADH subunit retains full enzyme activity [Hollocher and Place (1988) Genetics 116, 265-274]. Interestingly, the Ala159Thr mutant does not form either stable homodimers or heterodimers with wild-type ADH, suggesting that alanine-159 is important in stabilizing ADH dimers. The mutations were analysed in terms of a three-dimensional model of ADH using bacterial 20 beta-hydroxysteroid dehydrogenase and rat dihydropteridine reductase as templates. The model indicates that mutations in glycine-17 and glycine-93 affect the binding of NAD+. It also shows that alanine-159 is part of a hydrophobic anchor on the dimer interface of ADH. Replacement of alanine-159 with threonine, which has a larger side chain and can hydrogen bond with water, is likely to reduce the strength of the hydrophobic interaction. The three-dimensional model shows that glycine-184 is close to the substrate binding site. Replacement of glycine-184 with aspartic acid is likely to alter the position of threonine-186, which we propose hydrogen bonds to the carboxamide moiety of NAD+. Also, the negative charge on the aspartic acid side chain may interact with the substrate and/or residues in the substrate binding site. These mutations provide information about ADH catalysis and the stability of dimers, which may also be useful in understanding homologous dehydrogenases, which include the human 17 beta-hydroxysteroid, 11 beta-hydroxysteroid and 15-hydroxyprostaglandin dehydrogenases.

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.

scholarly journals Effects of Substrate-Binding Site Residues on the Biochemical Properties of a Tau Class Glutathione S-Transferase from Oryza sativa

Genes ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 25 ◽  
Author(s):  
Xue Yang ◽  
Jinchi Wei ◽  
Zhihai Wu ◽  
Jie Gao
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Abiotic Stress ◽  
Binding Site ◽  
Stress Responses ◽  
Stress Resistance ◽  
Substrate Binding ◽  
Biochemical Properties ◽  
Biotic And Abiotic Stress ◽  
Substrate Binding Site

Glutathione S-transferases (GSTs)—an especially plant-specific tau class of GSTs—are key enzymes involved in biotic and abiotic stress responses. To improve the stress resistance of crops via the genetic modification of GSTs, we predicted the amino acids present in the GSH binding site (G-site) and hydrophobic substrate-binding site (H-site) of OsGSTU17, a tau class GST in rice. We then examined the enzyme activity, substrate specificity, enzyme kinetics and thermodynamic stability of the mutant enzymes. Our results showed that the hydrogen bonds between Lys42, Val56, Glu68, and Ser69 of the G-site and glutathione were essential for enzyme activity and thermal stability. The hydrophobic side chains of amino acids of the H-site contributed to enzyme activity toward 4-nitrobenzyl chloride but had an inhibitory effect on enzyme activity toward 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide. Different amino acids of the H-site had different effects on enzyme activity toward a different substrate, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Moreover, Leu112 and Phe162 were found to inhibit the catalytic efficiency of OsGSTU17 to 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, while Pro16, Leu112, and Trp165 contributed to structural stability. The results of this research enhance the understanding of the relationship between the structure and function of tau class GSTs to improve the abiotic stress resistance of crops.

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