Mutational analysis of the interaction between active site residues and the loop region in mammalian purple acid phosphatases

Mammalian purple acid phosphatases (PAPs) can be divided into two groups, w hich exhibit distinct spectroscopic and kinetics properties: PAPs that cons ist of a single 36 kDa polypeptide, and PAPs that have undergone limited pr oteolysis to give two fragments with masses of 16 and 20 kDa, respectively. Proteolysis results in an increase in enzymatic activity, an increase in t he optimal pH for activity, and a change in the g(z) value of the character istic EPR spectrum of the mixed-valence binuclear iron center. It has been proposed that these changes are due to the loss of interactions between Asp 146 in an exposed loop region and active site residues upon proteolysis. In the present study, site-directed mutagenesis of Asp146 in recombinant rat bone PAP (recRPAP) has confirmed this hypothesis. Conversion of Asp146 into Ala, which eliminates the interaction of the side chain with the active si te, resulted in an enzyme with properties typical of PAPs isolated in prote olytically cleaved forms. The Asp146Asn and Asp146Glu mutants were also pre pared and examined to assess the effects of altered electrostatic interacti ons and side-chain length. Limited proteolysis of all three mutant enzymes with cathepsin L resulted in a significant increase in catalytic activity. Thus, although the interaction between Asp146 and (an) active site residue( s) is the major factor responsible for the low catalytic activity of unclea ved PAPs, other interactions are also important. Since both p-nitrophenyl p hosphate and osteopontin, a potential in vivo substrate, show the same leve l of activation, the observed increase in catalytic activity upon proteolys is is likely to be due to electrostatic rather than steric effects. EPR spe ctra of FeZn-recRPAP before and after cleavage by cathepsin L suggest that cleavage primarily affects the divalent metal site. The observation that pK (es,1) is also sensitive to changes at the divalent site is consistent with the proposal that the nucleophilic hydroxide is that bridging the divalent and trivalent metals.