CRYSTAL-STRUCTURE OF COMMON TYPE ACYLPHOSPHATASE FROM BOVINE TESTIS

Background: Acylphosphatase (ACP) is a low molecular weight phosphomon ohydrolase catalyzing with high specificity the hydrolysis of the carb oxyl-phosphate bond present in acylphosphates. The enzyme is thought t o regulate metabolic processes in which acylphosphates are involved, s uch as glycolysis and the production of ribonucleotides. Furthermore t he enzyme is capable of hydrolyzing the phospho-aspartyl intermediate formed during the action of membrane pumps such as (Ca2+ + Mg2+) ATPas e. Although the tertiary structure of a muscle ACP has been determined by NMR spectroscopy, little is known about the catalytic mechanism of ACP and further structures might provide an increased understanding. Results: The structure of 'common type' ACP from bovine testis has bee n determined by X-ray crystallography to a resolution of 1.8 Angstrom. The structure has been refined to an R factor of 17.0 % using all dat a between 15 and 1.8 Angstrom. The binding of a sulphate and a chlorid e ion in the active centre allows a detailed description of this site. The overall protein folds of common type and muscle ACP are similar b ut their loops have very different conformations. These differences, i n part, are probably caused by the binding of the ions in the active s ite of the common type form. The phosphate-binding loop of ACP shows s ome remarkable similarities to that of low molecular weight protein ty rosine phosphatase. Conclusions: The active site of ACP has been locat ed, enabling a reaction mechanism to be suggested in which the phospha te moiety bound to Arg23 acts as a base, abstracting a proton from a n ucleophilic water molecule liganded to Asn41. The transition-state int ermediate is stabilized by the phosphate-binding loop. We suggest the catalysis to be substrate assisted, which probably explains why this e nzyme can only hydrolyze acylphosphates.