The highly exposed loop region in mammalian purple acid phosphatase controls the catalytic activity.

Abstract

Recombinant human purple acid phosphatase (recHPAP) provides a convenient experimental system for assessing the relationship between molecular structure and enzymatic activity in mammalian purple acid phosphatases (PAPs). recHPAP is a monomeric protein with properties similar to those of uteroferrin (Uf) and other PAPs isolated as single polypeptide chains, but its properties differ significantly from those of bovine spleen PAP (BSPAP) and other PAPs isolated as proteolytically "clipped" forms. Incubation of recHPAP with trypsin results in proteolytic cleavage in an exposed region near the active site. The product is a tightly associated two-subunit protein whose collective spectroscopic and kinetics properties resemble those of BSPAP. These results demonstrate that the differences in spectroscopic and kinetics properties previously reported for mammalian PAPs are the result of proteolytic cleavage. Mass spectrometry shows that a three-residue segment, D-V-K, within the loop region is excised by trypsin. This finding suggests that important interactions between residues in the excised loop and one or more of the groups that participate in catalysis are lost or altered upon proteolytic cleavage. Analysis of available structural data indicates that the most important such interaction is that between Asp 146 in the exposed loop and active-site residues Asn 91 and His 92. Loss of this interaction should result in both an increase in the Lewis acidity of the Fe(II) ion and an increase in the nucleophilicity of the Fe(III)-bound hydroxide ion. Proteolytic cleavage thus constitutes a potential physiological mechanism for regulating the activity of PAP in vivo.