Identification of ATP citrate lyase as a phosphoprotein

Area of Science:

• Biochemistry
• Enzymology

Background:

• ATP citrate lyase (ACL) is crucial for fatty acid synthesis.
• Previous isolation methods often lead to protease degradation, yielding unstable enzyme preparations.
• Understanding ACL's structure and regulation is vital for metabolic studies.

Purpose of the Study:

• To develop a novel, high-yield purification procedure for intact and stable rat liver ATP citrate lyase.
• To characterize the purified enzyme, including its phosphorylation status and the nature of bound phosphate.
• To investigate the functional role of structural phosphate in ATP citrate lyase activity.

Main Methods:

• Purification of ATP citrate lyase from rat liver using a new procedure.
• Sodium dodecyl sulfate (SDS)-gel electrophoresis to assess enzyme purity and molecular weight.
• In vivo 32P-labeling of rat liver enzyme followed by purification.
• Enzymatic dephosphorylation using rat liver phosphatase and Vmax determination.

Main Results:

• A new procedure yielded highly pure, intact, and stable ATP citrate lyase (Mr = 110,000).
• Rat liver ATP citrate lyase is a phosphoprotein containing structural phosphate (serine phosphate) and catalytic phosphate.
• There are two structural phosphate residues per tetramer, located in a protease-sensitive region.
• Dephosphorylation did not alter the enzyme's Vmax, indicating structural phosphate is not essential for catalytic activity.

Conclusions:

• A robust method for purifying stable ATP citrate lyase has been established.
• Rat liver ATP citrate lyase is a phosphoprotein with distinct structural and catalytic phosphate groups.
• Structural phosphate, while present, does not influence the enzyme's maximal catalytic efficiency (Vmax).