Crystal structure of common type acylphosphatase from bovine testis

Abstract

The active site of ACP has been located, enabling a reaction mechanism to be suggested in which the phosphate moiety bound to Arg23 acts as a base, abstracting a proton from a nucleophilic water molecule liganded to Asn41. The transition-state intermediate is stabilized by the phosphate-binding loop. We suggest the catalysis to be substrate assisted, which probably explains why this enzyme can only hydrolyze acylphosphates.

Solution structure and conformational heterogeneity of acylphosphatase from Bacillus subtilis

“…Binding of phosphate is associated with marked stabilization of the active site loop 1 conformation, as the invisible NMR signals gradually appear, and the peak intensity of weak peaks gradually restore in this region. These observations further supports the idea that the cradle-like and ordered conformation of the active site loop observed in the X-ray structure is due to ligand binding [7]. The mobility of the active site loops as an important structure feature in ligand binding has been revealed in many phosphatases, such as low molecular weight protein tyrosine phosphatase from Bos taurus (PDB 1BVH) [26], Campylobacter jejuni (2GI4) [27], Tritrichomonas foetus (1P8A) [28], B. subtilis (1ZGG) [29], E. coli (2FEK) [30], and human PRL-3 (1V3Z) [31], and human phosphohistidine phosphatase 1 (2AI6) [32], suggesting that active site conformational flexibility should be considered to understand ligand recognition and design inhibitors.…”

“…However, some local differences are observed in the active site. In the crystal structure of CTAcP, loop 14-21 (loop 1) in the active site forms an ordered and cradle-like conformation [7]. However, in the solution structure of ligand-free BsAcP, the conformation of residues Gly14 to Arg18 in loop 1 is very flexible due to lack of distance constraints.…”

“…Structures of several AcPs from different organisms have been solved, most of which are in the ligand-bound state [6][7][8][9][10][11][12][13]. The first AcP (muscle type AcP) structure was determined by Nuclear magnetic resonance (NMR), and the structure in the ligand-free state revealed the presence of mobility in loop regions, although the overall RMS difference (RMSD) for the structure ensemble is rather high ($1.5 Å) [6,12,13].…”

The Contribution of Acidic Residues to the Conformational Stability of Common-Type Acylphosphatase

“…Structural studies based on NMR and X-ray crystallography (18,19), in combination with site-directed mutagenesis studies (32)(33)(34), have allowed the position of the active site to be located at atomic level. This includes the Arg23 and Asn41 residues and the 15-21 loop.…”

“…1 H NMR and X-ray crystallography have shown that both isoenzymes possess very similar threedimensional structures consisting of an antiparallel five-stranded ␤-sheet packed against two antiparallel ␣-helices ( Fig. 1) (18,19).…”