Diethyl pyrocarbonate inactivates CD39/ecto-ATPDase by modifying His-59.
Diethyl pyrocarbonate (DEPC) in conditions that favour carbethoxylation of histidyl residues strongly inactivated E-type ATPase activity of a rat lung membrane preparation, as well as ecto-ATPase activity of rat vessels and human Epstein-Barr virus-transformed B lymphocytes. Inactivation of the enzyme (up to 70%) achieved at concentrations of DEPC below 0.5 mM could be fully reversed by 200 mM hydroxylamine at pH 7.5, thus confirming histidine-selective modification. UTP effectively protected the enzyme activity from DEPC inactivation. This was taken to indicate that the conformation adopted by the enzyme molecule upon substrate binding was not compatible with DEPC reaching and/or modifying the relevant histidyl residue. Substrate activation curves were interpreted to show the enzyme molecule to be inactive, at all substrate concentrations tested, when the target histidyl residue had been modified by DEPC. Comparison of known sequences of CD39-like ecto-ATP(D)ases with the results on inactivation by DEPC revealed His-59 and His-251 (according to the human CD39 sequence) as equally possible targets of the inactivating DEPC modification. Potato apyrase lacks a homologue for the former residue, while the latter is preserved in the enzyme sequence. Therefore, this enzyme was exposed to DEPC, and since hydrolysis of ATP and ADP by potato apyrase was insensitive to modification with DEPC, it was concluded that His-59 is the essential residue in CD39 that is affected by DEPC modification in a way that causes inactivation of the enzyme.
Dzhandzhugazyan
K N
KN
Institute of Cancer Biology, Danish Cancer Society, Copenhagen.
Plesner
L
L
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002461:Cell Line, Transformed; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006639:Histidine / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D051381:Rats; D017386:Sequence Homology, Amino Acid
D004791:Enzyme Inhibitors; D006639:Histidine; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D004047:Diethyl Pyrocarbonate