STUDIES ON THE CATALYTIC MECHANISM OF PIG PURPLE ACID-PHOSPHATASE

Several independent experiments failed to reveal any evidence in suppo rt of the involvement of a phosphoryl-enzyme intermediate in the catal ytic mechanism of pig allantoic fluid purple acid phosphatase: (i) att empts to label enzyme with phosphate derived from [P-32]p-nitrophenyl phosphate were unsuccessful; (ii) values of k(cat) for a series of pho sphate derivatives varied over a wide range, with the enzyme showing a marked preference for activated ester and anhydride substrates over t hose with a stable leaving group; (iii) burst titrations revealed a '' burst'' of p-nitrophenol from p-nitrophenyl phosphate only when the en zyme was added after the substrate, suggesting that this result was an artifact of the order of addition of reagents; (iv) transphosphorylat ion from p-nitrophenyl phosphate to acceptor alcohols could not be det ected, even under conditions where a transphosphorylation to hydrolysi s ratio as low as 0.015 could have been measured; (v) enzyme-catalyzed exchange of O-18 between phosphate and water was demonstrated, althou gh at a rate much slower than that observed for other phosphatases whe re the involvement of a phosphoryl-enzyme intermediate in the mechanis m has been clearly established. The present results are compared with those obtained in similar studies on other phosphatases, particularly the highly homologous beef spleen purple acid phosphatase, and their i mplications for the catalytic mechanism of the purple acid phosphatase s are discussed. (C) 1995 Academic Press, Inc.