Histidine, diethylpyrocarbonate

Similarly, the rate of inhibition of phosphoenzyme formation by diethylpyrocarbonate (DEPC) was much slower than the loss of ATPase activity [368], Even when the reaction approached completion with more than 90% inhibition of ATP hydrolysis, about 70% of the Ca -ATPase could still be phosphorylated by ATP (2.3nmoles of E P/mg protein). The remaining 30% of E P formation and the corresponding ATPase activity was not reactivated by hydroxylamine treatment, suggesting some side reaction with other amino acids, presumably lysine. When the reaction of the DEPC-modified ATPase with P-ATP was quenched by histidine buffer (pH 7.8) the P-phosphoenzyme was found to be exceptionally stable under the same conditions where the phosphoenzyme formed by the native ATPase underwent rapid hydrolysis [368]. The nearly normal phosphorylation of the DEPC-trea-ted enzyme by P-ATP implies that the ATP binding site is not affected by the modification, and the inhibition of ATPase activity is due to inhibition of the hydrolysis of the phosphoenzyme intermediate [368]. This is in contrast to an earlier report by Tenu et al. [367], that attributed the inhibition of ATPase activity by... 

Dicyanocobalamin 870 3,6-Dideoxyhexoses, formation of 747 Dielectric constant 47 Diesenhofer, Johan 84 Diethanolamine, pKa value of 99 Diethylmalonic acid, pKa value of 99 Diethylpyrocarbonate reaction with histidine 126... 

Modified histidine residues of HRP elucidated by the specific chemical modification of histidine residues of CPO with diethylpyrocarbonate The catalytic activity of HRP was not [60]... 

The structure and enzyme kinetics of bovine erythrocyte superoxide dismutase are reviewed. The protein has a novel imidazolate-bridged copper(II)-zinc(II) catalytic center in each of two identical subunits. Since a C /Cu1 redox couple is responsible for the dismutase activity of the enzyme, the role of zinc is of interest. Both 220-MHz NMR measurements of the exchangeable histidine protons and chemical modifications using diethylpyrocarbonate demonstrate that zinc alone can fold the protein chain in the region of the active site into a conformation resembling that of the native enzyme. Other possible roles for zinc are discussed. Synthetic, magnetic, and structural studies of soluble, imidazolate-bridged copper complexes of relevance to the 4 Cu(II) form of the enzyme have been made. 

The extreme sensitivity of RNA to the ubiquitous inter- and intracellular nudeases (for example on the skin of the investigator) makes special precautions necessary for effective RNA preparations. The use of disposable containers is recommended, or glassware that has been soaked in dilute hydrochloric acid and rinsed with autoclaved distilled water. Disposable gloves must be worn in all procedures where RNA is handled, or is likely to come into contact with RNA, such as solutions, chemicals, glassware, spatulas etc. Buffers for RNA work should be prepared from reagents reserved for this purpose, and stored separately. Buffers can be treated with 0.2 % (v/v) diethylpyrocarbonate (care - this is carcinogenic) and autodaved to inactivate RNases, or at least those with adive site histidines. Since most nucleases require Mg2 for activity, the addition of EDTA in mM concentrations to solutions is also recommended. 

Histidine residues in the horse enzyme have also been modified by carbethoxylation with diethylpyrocarbonate. This was found to result in a rapid activation followed by a slower inactivation (160). Substitution of the enzyme was measured, but affected histidine residues were not identified. They were, however, suggested to be outside the active site. 

The activity of arsenite oxidoreductase from A. faecalis is affected by essential histidines (43). Approximately three histidine residues in the oxidized enzyme are readily accessible to chemical modification by diethylpyrocarbonate, and at least one of these modulates the activity of the oxidized enzyme. However, if arsenite oxidoreductase is first reduced by either dithionite (a low potential generic reductant) or by arsenite, approximately three histidines can be modified, without affecting arsenite oxidoreductase activity. The reductive half reaction of arsenite oxidoreductase may therefore be dependent on histidine residue(s) either for the process of electron transfer or for the correct conformation of the oxidized protein. As indicated above. His 195 and His423 form part of the binding site and one of these may be the residue whose modification in oxidized enzyme results in loss of activity. 

Cells of Scenedesmus obliauus (wild type) were grown heterotrophically in the dark. PS II membranes were prepared as described in [7]. The following proteases were used to modify the PS II membranes carboxypeptidase A, 1 h incubation with 1 2 Chhenzyme subtilisin Carlsberg, 30 min incubation with 20 1 Chhenzyme and trypsin, 10 min incubation with 50 1 Chhenzyme. Incubation with trypsin was performed at pH 7.4, otherwise all treatments were done at pH 6.5. The reactions were stopped by 20 times dilution with ice-cold 20 mM MES-NaOH pH 6.5, 20 mM NaCl, 400 mM sucrose, and 1 mM phenylmethylsulfonyl fluoride (PMSF), except for carboxypeptidase A incubations where 4 mM 1,10-phrenanthroline was substituted for PMSF. Mn depletion of PS II membranes was accomplished by a 30 min incubation with either 0.8 M Tris-HCl pH 8.4 or 5 mM NHjOH pH 6.5. Histidine and carboxyl residues in PS II membranes were modified with 500 /xM diethylpyrocarbonate (DEPC) [8] or 10 mM l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) [9], respectively. More details regarding these procedures can be found in [5]. 

Purified cottonseed NAPE synthase enzyme exhibited non-Michaelis-Menten biphasic kinetics with respect to the free fatty acid substrates, palmitic and linoleic acids. Kinetic parameters for the two saturable sites were calculated from various transformations e.g., double-reciprocal and Hill plots Cornish-Bowden, 1995) of initial velocity/ substrate concentration data and are summarized in TABLE 1. Preliminary experiments with several group-specific modifiers indicated that NAPE synthase was progressively inactivated by increasing concentrations of 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB), diisopropyl fluorophosphate (DFP), phenylmethylsulfonylfluoride (PMSF), diethylpyrocarbonate (DEPC) (TABLE 2). These results suggest that NAPE synthase may form a thioester- or ester-intermediate through a cysteine or serine residue, respectively, and a histidine residue may participate in catalysis as well. 

Diethylpyrocarbonate (DEPC) is a broad and highly sensitive histidine modifier. DEPC reacts with His residues in proteins to yield an N-carbethoxyhistidyl derivative, followed by an increase in absorbance at 240 nm [121]. The reaction is as follows ... 

Ovadi J, Libor S and Elodi P (1967) Spectophotometric determination of histidine in proteins with diethylpyrocarbonate, Acta Biochim. Biophys. 

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