Diethylpyrocarbonate reaction with histidin

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... 

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... 

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]. 

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 ... 

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