Substrate specificity of Gaucher spleen phosphoprotein phosphatase

The spleen in Gaucher's disease contains elevated levels of two distinct acid phosphatases. One of the isoenzymes, a tartrate-resistant type 5 acid phosphatase which we have designated SP_II acid phosphatase, possesses considerable phosphoprotein phosphatase activity. The enzyme dephosphorylates phosvitin and casein at specific rates (V) of 38.6 and 45.0 units/mg, respectively. The dephosphorylation of the oligophosphoproteins as well as various fragments of phosvitin, histories, and monophosphopeptides was studied kinetically. Positive cooperativity (Hill coefficient = 1.3–2.0) was observed for the dephosphorylation of phosvitin and casein as well as for the dephosphorylation of fragments of phosvitin which contained as few as two vicinal phosphoserine residues. In contrast, the hydrolysis of phosphomonoesters such as o-phosphorylserine or various monophosphopeptides exhibited typical Michaelis-Menten kinetics. Cooperativity appears to depend upon the substrate rather than the enzyme. The cooperativity of dephosphorylation was not affected by altering the secondary structure of phosvitin from a random to β conformation or by acetylation of the protein; however, acetylated phosvitin was dephosphorylated more rapidly (V = 50.8 units/mg) than native phosvitin indicating that the very basic phosphatase enzyme (pI = 8.5) prefers more acidic phosphoproteins as substrates rather than basic proteins such as histone (V= 0.0013 unit/mg). A monophosphohexa-peptide (V = 0.47 unit/mg) and monophosphoheptapeptide (V = 0.18 unit/mg) proved to be much poorer substrates than phosvitin, and monophosphoproteins such as glycogen phosphorylase, phosphorylase kinase, and glycogen synthase were not dephosphorylated by the enzyme. Although the phosphatase is active on monophosphopeptides and the presence of flanking amino acids considerably decreases the K_m of the enzyme for the phosphoserine residue (up to 100-fold), the enzyme appears to prefer peptide or protein substrates that contain two or more phosphoserine residues in close proximity. Finally, previous results showing the spleen phosphatase to be composed of 16,000- and 20,000-dalton subunits were apparently due to proteolysis during isolation since when 1.0 mm phenylmethylsulfonyl fluoride was included in the isolation media, the enzyme appeared as a single 35,000-dalton species when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.