Diethyl pyrocarbonate inactivates CD39/ecto-ATPDase by modifying His-59

Area of Science:

• Biochemistry
• Enzymology

Background:

• E-type ATPase and ecto-ATPases are crucial enzymes involved in cellular energy metabolism and signaling.
• Histidyl residues are known to play important roles in enzyme active sites.
• Diethyl pyrocarbonate (DEPC) is a chemical reagent used to modify histidine residues.

Purpose of the Study:

• To investigate the role of histidyl residues in the activity of E-type ATPase and related ecto-ATPases.
• To identify specific histidyl residues critical for enzyme function using DEPC as a chemical probe.

Main Methods:

• Enzyme activity assays using rat lung membrane preparations, rat vessels, and human lymphocytes.
• Chemical modification of enzymes with DEPC under varying conditions.
• Reversal of inactivation using hydroxylamine.
• Protection studies using UTP.
• Sequence analysis of CD39-like ecto-ATPases and potato apyrase.

Main Results:

• DEPC strongly inactivated E-type ATPase and ecto-ATPase activities.
• Inactivation was reversible with hydroxylamine, confirming histidine modification.
• UTP protected enzyme activity, indicating substrate-induced conformational changes.
• Sequence analysis pointed to His-59 and His-251 in CD39 as potential targets.
• Potato apyrase, lacking His-59, was insensitive to DEPC, implicating His-59 in CD39 inactivation.

Conclusions:

• Histidine modification by DEPC is responsible for the inactivation of E-type ATPase and CD39 ecto-ATPases.
• His-59 in CD39 is an essential residue for enzyme activity, and its modification by DEPC leads to inactivation.
• Substrate binding alters enzyme conformation, preventing DEPC modification of the critical histidine residue.