Investigation of the Methanosarcina thermophila acetate kinase mechanism by fluorescence quenching.

Acetate kinase [E.C. 2.7.2.1] is a homodimer which catalyzes the reversible magnesium-dependent transfer of the γ-phosphoryl group from ATP to acetate, yielding ADP and acetyl phosphate (CH3COO- + ATP ⇔ CH3CO2PO3-2 + ADP). The enzyme is widely distributed among fermentative prokaryotes in the Bacteria domain where it functions together with phosphotransacetylase (CH3CO2PO3-2 + HS-CoA ⇔ CH3COSCoA + HPO4-2) to convert acetyl-CoA to acetate and synthesize ATP. These enzymes also function to activate acetate to acetyl-CoA in the first step of the pathway for conversion of the methyl group to methane by Methanosarcina species from the Archaea domain (1). Though acetate kinase was one of the earliest phosphoryl transfer enzymes to be identified (2) and investigated, a debate persisted in the literature until recently whether the catalytic mechanism is a triple displacement of the phosphate involving two covalent enzyme intermediates (3) or a direct in-line phosphoryl transfer (4, 5). Support for the direct in-line mechanism accrues from studies with the acetate kinase from Methanosarcina thermophila (6-12). The enzyme co-crystallized with ADP, Al+3, F-, and acetate, shows a linear array of ADP-AlF3-acetate in the active site cleft wherein the AlF3 is proposed to mimic the meta-phosphate transition state (11). Kinetic analyses of Arg241 and Arg91 replacement variants indicate these active site residues are essential for catalysis and important for binding acetate (11). In a direct in-line phosphoryl transfer, the hypothesized transition state is a trigonal bipyramidal structure of the γ-phosphoryl group, with the axial positions occupied by β-phosphoryl and acetyl oxygens. The guanidino groups of Arg241 and Arg91 are proposed to stabilize this structure by interacting with oxygen atoms of the γ-phosphoryl and carboxyl groups of acetate (11). The guanidino group of Arg241 is adjacent to AlF3 consistent with the proposed role. However, the guanidino group of Arg91 is displaced 7A from AlF3 placing doubt on the proposed role, although the structure may not accurately represent the position of Arg91 during catalysis (11). Based upon structural similarities of the M. thermophila enzyme, acetate kinase was identified as a member of the acetate and sugar kinase/Hsc70/actin (ASKHA) structural superfamily (12). Each monomer of the homodimeric acetate kinase is characterized by a duplicated central β-sheet surrounded by α-helices (βββαβαβα core) and consisting of two domains (Fig. 1). The sugar kinase and actin family members are all known to undergo a catalytically essential domain closure upon ligand binding (13-18). Although there is no experimental evidence to support domain closure during catalysis of acetate kinase from M. thermophila, domain closure has been suggested to participate in stabilization of the transition state based upon the architecture of the crystal structure (12). Figure 1 A. Structure of the M. thermophila acetate kinase (1TUY, (11)). The dimer is shown with monomer A (ribbon representation) in rainbow hue from the blue N-terminus to the red C-terminus. Monomer B (wire representation) with domain I (residues 1-148 and ... The M. thermophila acetate kinase has no tryptophan residues which has precluded intrinsic fluorescence measurements as an approach to investigate the catalytic mechanism. Here we report Kd values for ADP:Mg and ATP:Mg for the M. thermophila acetate kinase enabled by fluorescence quenching of the Gln43Trp variant. Double variants, with either Arg241 or Arg91 replaced in the Gln43Trp variant, had Kd values consistent with previously proposed roles for these arginines in catalysis and substrate binding. The Gln43Trp variant has also provided evidence for domain motion with implications for the catalytic mechanism. The results indicate that catalysis is not as two independent active sites, but that the active site activities are coordinated in a half-the-sites manner.