title: Effect of limited proteolysis on phospholipase C-gamma1 kinetics.
abstract: Phospholipase C-gamma1 is a tightly regulated, multidomain protein that generates the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. Kinetic analysis reveals that phospholipase C-gamma1 displays apparent allosteric behavior. A previous study determined that proteolytic cleavage of the SH domain region of phospholipase C-gamma1 yields an activated form of the enzyme (A. W. Fernald, G. A. Jones, and G. Carpenter Biochem. J. 302, 508, 1994). In this study, we show that activation of phospholipase C-gamma1 by proteolysis decreases both the cooperativity and the half-maximal value of the enzyme for substrate. Kinetic analysis revealed that the mole fraction of phosphatidylinositol 4,5-bisphosphate (PIP(2)) that resulted in half-maximal PIP(2) hydrolysis (S(0.5)) was lower for proteolyzed than uncleaved phospholipase C-gamma1 (0.08 mole fraction vs 0.18 mole fraction of PIP(2)). The cooperativity index was lower for proteolyzed than full-length phospholipase C-gamma1 (n = 2.5 vs n = 4). Kinetic analysis also revealed that the estimated dissociation constant was lower for phospholipase C-gamma1 that had been subjected to proteolysis (0.1 mM vs 1.0 mM PIP(2) for cleaved vs uncleaved phospholipase C-gamma1, respectively). It was previously hypothesized that activation of phospholipase C-gamma1 requires a conformational change that results in increased accessibility of substrate to the active site and that the SH domain of the enzyme is involved in the activation event. These experiments support the hypothesis that a portion of the protein covers the active site, allosterically inhibiting the enzyme, and that the removal of this "lid" domain activates the enzyme.
authors.affiliation: Department of Pharmacology, University of Virginia Health Sciences Center, Charlottesville, Virginia, 22908, USA. gaj5m@virginia.edu
authors.forename: G A
authors.initials: GA
authors.lastname: Jones
authors.forename: Y
authors.initials: Y
authors.lastname: Wu
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D015153:Blotting, Western; D004789:Enzyme Activation; D006868:Hydrolysis; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008823:Micelles; D008958:Models, Molecular; D008970:Molecular Weight; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D051966:Phospholipase C gamma; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000378:metabolism*; D013816:Thermodynamics; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism*; D018909:src Homology Domains
chemical_list: D007527:Isoenzymes; D008823:Micelles; D019269:Phosphatidylinositol 4,5-Diphosphate; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase