Mechanism of phosphatidylinositol-specific phospholipase C: Origin of unusually high nonbridging thio effects

Phosphatidylinositol-specific phospholipase C (PI-PLC) has been proposed pr eviously to employ a catalytic mechanism highly reminiscent of that of ribo nuclease A (RNase A). Both catalytic sites are comprised of two histidine s ide chains acting as a general base-general acid pair and a phosphate-activ ating residue: an arginine in the case of PI-PLC and a lysine in RNase A. D espite these structural similarities, the PI-PLC reaction is slowed 10(5)-f old upon substitution of one of the phosphate nonbridging oxygen atoms with sulfur, whereas a much smaller effect is observed in the analogous RNase A reaction. Here, we report a systematic study of this property in PI-PLC, c onducted by means of site-directed chemical modification of a cysteine resi due replacing the arginine at position 69. The results show that mutant enz ymes featuring bidentate side chains at this position display significantly higher activity, higher thio effects, and greater stereoselectivity than d o those with monodentate side chains. The results suggest that the bidentat e nature of Arg69 is the origin of the large thio effects and stereoselecti vity in PI-PLC. We propose that in addition to binding the phosphate, the f unction of arginine 69 is to bring the phosphate group and the 2-OH group o f inositol into proximity and to induce proper alignment for nucleophilic a ttack, and possibly to lower the pK(a) of the 2-OH. The results presented h ere could be important to mechanisms of phosphoryl transfer enzymes in gene ral, suggesting that a major part of thio effects observed in enzymatic pho sphoryl transfer reactions can originate from factors other than direct int eraction between a side chain and a phosphate group, and caution the use of the absolute magnitude of the thio effect as an indicator of the strength of such interactions.