Phosphatidylinositol-specific phospholipase C cyclic phosphodiesterase activity depends on solvent polarity.
Large enhancements (maximum of 82-fold in terms of enzyme efficiency, Vmax/Km) of bacterial PI-PLC cyclic phosphodiesterase activity were observed in the presence of organic solvents miscible in water (dimethyl sulfoxide, dimethylformamide, and 2-propanol). In general, organic solvents lowered the Km for myo-inositol 1,2-cyclic phosphate (cIP) and increased Vmax substantially. This kinetic effect was similar to that obtained with phosphatidylcholine micelles and bilayers in an aqueous assay system for cyclic inositol phosphate hydrolysis [Zhou, C., et al. (1997) Biochemistry 36, 347-355]. Solvent properties were examined to determine which ones correlated with the activation of PI-PLC toward cIP in each solvent. Activation correlated best with the solvent polarity as measured by ET(30); no significant correlation was observed with solution surface tension, the bulk dielectric constant (epsilon), 1/epsilon (a measure of the strength of charge interactions), or the Hildebrand solubility parameter. The sigmoidal curve of the enzyme activity versus solvent polarity was consistent with the solvent promoting a transition in the enzyme from a low-activity to a high-activity form. Possible candidates for this change, including enzyme dimerization, helix B/loop stabilization, and dehydration of the active site, are discussed.
Wu
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Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02167, USA.
Roberts
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D000433:1-Propanol / Q000494:pharmacology; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001413:Bacillus thuringiensis / Q000201:enzymology / Q000235:genetics; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004126:Dimethylformamide / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism*; D007700:Kinetics; D015394:Molecular Structure; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D011487:Protein Conformation; D012997:Solvents / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013500:Surface Tension; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism*
D007295:Inositol Phosphates; D012997:Solvents; C010199:inositol cyclic phosphate; D004126:Dimethylformamide; D000433:1-Propanol; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D004121:Dimethyl Sulfoxide