Investigating the mechanism of ADP-forming acetyl-CoA synthetase from the protozoan parasite Entamoeba histolytica

Abstract

ADP-forming acetyl-CoA synthetase (ACD) catalyzes the interconversion of acetyl-CoA and acetate. The related succinyl-CoA synthetase follows a three-step mechanism involving a single phosphoenzyme, but a novel four-step mechanism with two phosphoenzyme intermediates was proposed for Pyrococcus ACD. Characterization of enzyme variants of Entamoeba ACD in which the two proposed phosphorylated His residues were individually altered revealed that only His252 is essential for enzymatic activity. Analysis of variants altered at two residues proposed to interact with the phosphohistidine loop that swings between distinct parts of the active site are consistent with a mechanism involving a single phosphoenzyme intermediate. Our results suggest ACDs with different subunit structures may employ slightly different mechanisms to bridge the span between active sites I and II.

Keywords: ATP; Entamoeba; acetate; acetyl-CoA; acetyl-CoA synthetase; energy metabolism.

FIGURE 1

Comparison of ACD and SCS. (A) Schematic of the domain organization in ACD and SCS. Each is comprised of five domains with relative sizes as shown arranged in a unique pattern. ACD enzymes may have a hinge region (indicated by the black rectangle) that connects the alpha and beta domains into a single subunit as opposed to separate α and β subunits. SCS enzymes are encoded as two separate subunits as shown. (B) Sequence alignment of E. coli SCS and ACD from multiple organisms. α and β indicate the respective subunit for those enzymes in which the alpha and beta domains are encoded by separate genes.

FIGURE 2

Activity of EhACD variants. Activity was determined in the acetyl-CoA forming direction (light gray) and in the acetate-forming direction (dark gray). Activities are represented as a percentage of the activity observed for the wild-type enzyme (100%). Assays were performed in triplicate and error bars represents standard deviation. (A) Activity of the His252 and His533 variants. (B) Activity of the Glu213 and Asp674 variants.

FIGURE 3

Isotopic labeling of EhACD. Enzymes were labeled with ³²P_i + acetyl-CoA (top panels) or with [γ-³²P]ATP (bottom panels). Assays were performed in triplicate. (A) Analysis of the His252Ala and His533Ala variants versus wild-type enzyme. (B) Analysis of the Glu213Ala and Asp674Ala variants versus wild-type enzyme.

FIGURE 4

Distance measurements in Site II of CKcACDI. The structure of CKcACDI crystallized with CoA and MgAMPPCP (PDB accession #4XZ3) showing Hisα in Site II of the active site was used for distance measurements. His254α is shown in purple and His68β is shown in blue. MgAMPPCP is colored by element: C (gray), P (orange), O (red), N (purple), H (light gray), and Mg (green). The distance between N3 of Hisα and P of AMPPCP (yellow line) is 5.7 Å. The distances between Hisα and Hisβ (orange line) and between Hisβ and P of AMPPCP (green line) are 9 Å and 7.9 Å, respectively.