Mutation of two active-site residues converts a phosphatidylinositol-specific phospholipase C to a glucose phosphatase.
Two mutations, R69D and K115E, converted a bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) to a phosphatase with much higher specific activity toward glucose-6-phosphate than inositol-1-phosphate. PI-PLC single mutations R69D and K115E can cleave PI but lack any demonstrable phosphatase activity. The bacterial PI-PLC has no sequence homology with known glucose-6-phosphatase enzymes, which need His, Arg, and negatively charged residues (Asp or Glu) at the active site. The change in chemical reaction and substrate specificity can be rationalized by energy minimization of the mutant with I-1-P or G-6-P bound.
Feng
Jianwen
J
Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, USA.
Stieglitz
Kimberly
K
Roberts
Mary F
MF
D019943:Amino Acid Substitution; D001665:Binding Sites; D005952:Glucose-6-Phosphatase / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D043265:Phosphatidylinositol Diacylglycerol-Lyase / Q000235:genetics* / Q000378:metabolism*; D054801:Phosphoinositide Phospholipase C; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D011994:Recombinant Proteins; D005952:Glucose-6-Phosphatase; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase