The structure of succinyl-CoA synthetase bound to the succinyl-phosphate intermediate clarifies the catalytic mechanism of ATP-citrate lyase

Succinyl-CoA synthetase (SCS) catalyzes a three-step reaction in the citric acid cycle with succinyl-phosphate proposed as a catalytic intermediate. However, there are no structural data to show the binding of succinyl-phosphate to SCS. Recently, the catalytic mechanism underlying acetyl-CoA production by ATP-citrate lyase (ACLY) has been debated. The enzyme belongs to the family of acyl-CoA synthetases (nucleoside diphosphate-forming) for which SCS is the prototype. It was postulated that the amino-terminal portion catalyzes the full reaction and the carboxy-terminal portion plays only an allosteric role. This interpretation was based on the partial loss of the catalytic activity of ACLY when Glu599 was mutated to Gln or Ala, and on the interpretation that the phospho-citryl-CoA intermediate was trapped in the 2.85   Å resolution structure from cryogenic electron microscopy (cryo-EM). To better resolve the structure of the intermediate bound to the E599Q mutant, the equivalent mutation, E105 α Q, was made in human GTP-specific SCS. The structure of the E105 α Q mutant shows succinyl-phosphate bound to the enzyme at 1.58   Å resolution when the mutant, after phosphorylation in solution by Mg2+-ATP, was crystallized in the presence of magnesium ions, succinate and desulfo-CoA. The E105 α Q mutant is still active but has a specific activity that is 120-fold less than that of the wild-type enzyme, with apparent Michaelis constants for succinate and CoA that are 50-fold ...

Source: Acta Crystallographica Section F - September 26, 2022 Category: Biochemistry Authors: Huang, J. Fraser, M.E. Tags: succinyl-CoA synthetase succinyl-phosphate cryogenic electron microscopy catalytic intermediates ATP-citrate lyase research communications Source Type: research