Action of phosphatidylinositol-specific phospholipase C gamma 1 on solubleand micellar substrates - Separating effects on catalysis from modulation of the surface

(T)he kinetics of PI-PLC gamma 1 toward a water-soluble substrate (inositol 1,2-cyclic phosphate, cIP) and phosphatidylinositol (PI) in detergent mixe d micelles were monitored by P-31 NMR spectroscopy. That cIP is also a subs trate (K-m = similar to 15 mM) implies a two-step mechanism (intramolecular phosphotransferase reaction to form cIP followed by cyclic phosphodiestera se activity to form inositol-1-phosphate (I-1-P)), PI is cleaved by PI-PLC gamma 1 to form cIP and I-I-P with the enzyme specific activity and ratio o f products (cIP/I-1-P) regulated by assay temperature, pH, Ca2+, and other amphiphilic additives, Cleavage of both cIP and PI by the enzyme is optimal at pH 5, The effect of Ca2+ on PI-PLC gamma 1 activity is unique compared with other isozymes enzymes: Ca2+ is necessary for the activity and low Ca2 + activates the enzyme; however, high Ca2+ inhibits PI-PLC gamma 1 hydrolys is of phosphoinositides (but not cIP) with the extent of inhibition depende nt on pH, substrate identity (cIP or PI), substrate presentation (e.g. dete rgent matrix), and substrate surface concentration. This inhibition of PI-P LC gamma 1 by high Ca2+ is proposed to derive from the divalent metal ion-i nducing clustering of the PI and reducing its accessibility to the enzyme, Amphiphilic additives such as phosphatidic acid, fatty acid, and sodium dod ecylsulfate enhance PI cleavage in micelles at pH 7.5 but not at pH 5.0; th ey have no effect on cIP hydrolysis at either pH value. These different kin etic patterns are used to propose a model for regulation of the enzyme. A k ey hypothesis is that there is a pH-dependent conformational change in the enzyme that controls accessibility of the active site to both water-soluble cIP and interfacially organized PI. The low activity enzyme at pH 7.5 can be activated by PA (or phosphorylation by tyrosine kinase), However, this a ctivation requires lipophilic substrate (PI) present because cIP hydrolysis is not enhanced in the presence of PA.