Unique catalytic mechanism of phosphatidylinositol-specific phospholipase C from Streptomyces antibioticus.
Calcium-dependent phosphatidylinositol-specific phospholipase C from Streptomyces antibioticus (saPLC1) catalyzes hydrolysis of phosphatidylinositol (PI) into inositol 1-phosphate by a unique mechanism involving formation of inositol 1,6-cyclic phosphate (1,6-IcP) as an intermediate. This work examines the rates and products of cleavage of phosphorothioate and phosphorodithioate analogues of PI in which sulfur was introduced into the phosphate moiety at a nonbridging position (pro-R or pro-S), a bridging position, or both. The replacement of the pro-S oxygen in the phosphoryl moiety of PI by sulfur results in a 3 x 10(7)-fold decrease of the catalytic rate constant, whereas alteration of the pro-R oxygen results in only a modest rate reduction. The addition of the second sulfur atom into the bridging position of the S(p) isomer of the phosphorothioate analogue causes a dramatic (2 x 10(5)-fold) increase of the rate of cleavage but has a negligible effect on the R(p) isomer. These differences are consistent with a change in the mechanism for the S(p) isomer of the phosphorodithioate analogue into a more dissociative type, where the leaving group carries a large amount of negative charge. In addition, hydrolysis of the diastereomers of the phosphorothioate analogues of 1,6-IcP, inositol cis-1,6-IcPs and inositol trans-1,6-IcPs, affords two distinct products, inositol 1-phosphorothioate and inositol 6-phosphorothioate, respectively. Formation of inositol 6-phosphorothioate is explained by the binding of trans-1,6-IcPs in the active site in a rotated orientation that interchanges the oxygen atoms at the 1- and 6-positions, thereby allowing the hydroxyl group at the 1-position to act as a leaving group. The reorientation of the intermediate is driven by formation of favorable interactions of the enzyme active site with the nonbridging oxygen in the trans intermediate.
Bai
Chuan
C
Department of Medicinal Chemistry and Pharmacognosy, The University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Zhao
Li
L
Tsai
Ming-Daw
MD
Bruzik
Karol S
KS
D007700:Kinetics; D015394:Molecular Structure; D010710:Phosphates / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C / Q000378:metabolism*; D013303:Streptomyces antibioticus / Q000201:enzymology*; D013455:Sulfur / Q000378:metabolism
D010710:Phosphates; D010716:Phosphatidylinositols; D013455:Sulfur; C083120:phosphorodithioic acid; D054801:Phosphoinositide Phospholipase C