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

Biochemistry
A V KravchukM D Tsai

Abstract

Phosphatidylinositol-specific phospholipase C (PI-PLC) has been proposed previously to employ a catalytic mechanism highly reminiscent of that of ribonuclease A (RNase A). Both catalytic sites are comprised of two histidine side chains acting as a general base-general acid pair and a phosphate-activating residue: an arginine in the case of PI-PLC and a lysine in RNase A. Despite these structural similarities, the PI-PLC reaction is slowed 10(5)-fold 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, conducted by means of site-directed chemical modification of a cysteine residue replacing the arginine at position 69. The results show that mutant enzymes featuring bidentate side chains at this position display significantly higher activity, higher thio effects, and greater stereoselectivity than do those with monodentate side chains. The results suggest that the bidentate nature of Arg69 is the origin of the large thio effects and stereoselectivity in PI-PLC. We propose that in addition to binding the phosphate, the function of arginine 69 is to bring the phosphat...Continue Reading

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Citations

Apr 17, 2010·Amino Acids·Robert J Hondal, Erik L Ruggles
Jan 31, 2006·Journal of the American Society for Mass Spectrometry·Keiichiro OharaJean-Jacques Vasseur
May 9, 2003·Bioorganic & Medicinal Chemistry·Yinghui LiuWonhwa Cho
Apr 22, 2009·Journal of the American Chemical Society·Jason M ThomasDavid M Perrin
Nov 6, 2012·Antioxidants & Redox Signaling·Robert J HondalVadim N Gladyshev
Jul 5, 2003·Biological & Pharmaceutical Bulletin·Takashi ObamaKikuo Tsukamoto
May 2, 2009·Biochimica Et Biophysica Acta·Robert J Hondal
Mar 13, 2003·Journal of the American Chemical Society·Cornelia MihaiKarol S Bruzik

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