Glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) specifically cleaves GPIs. This phospholipase D is a secreted protein consisting of two domains: an N-terminal catalytic domain and a predicted C-terminal β-propeller. Although the biochemical properties of GPI-PLD have been extensively studied, its catalytic site has not been identified. We hypothesized that a histidine residue(s) may play a critical role in the catalytic activity of GPI-PLD, based on the observations that (i) Zn2+, which utilizes histidine residues for binding, is required for GPI-PLD catalytic activity, (ii) a phosphohistidine intermediate is involved in phospholipase D hydrolysis of phosphatidylcholine, (iii) computer modelling suggests a catalytic site containing histidine residues, and (iv) our observation that diethyl pyrocarbonate, which modifies histidine residues, inhibits GPI-PLD catalytic activity. Individual mutation of the ten histidine residues to asparagine in the catalytic domain of murine GPI-PLD resulted in three general phenotypes: not secreted or retained (His56 or His88), secreted with catalytic activity (His34, His81, His98 or His219) and secreted without catalytic activity (His29, His125, His133 or His158). Changing His133 but not His29, His125 or His158 to Cys resulted in a mutant that retained catalytic activity, suggesting that at least His133 is involved in Zn2+ binding. His133 and His158 also retained the biochemical properties of wild-type GPI-PLD including trypsin cleavage pattern and phosphorylation by protein kinase A. Hence, His29, His125, His133 and His158 are required for GPI-PLD catalytic activity.
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Research Article|
October 10 2005
Mutating His29, His125, His133 or His158 abolishes glycosylphosphatidylinositol-specific phospholipase D catalytic activity
Nandita S. Raikwar;
Nandita S. Raikwar
1Department of Medicine and Department of Biochemistry and Molecular Biology, Indiana University School of Medicine and the Department of Veterans Affairs, Richard L. Roudebush VAMC, Indianapolis, IN 46202, U.S.A.
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Rosario F. Bowen;
Rosario F. Bowen
1Department of Medicine and Department of Biochemistry and Molecular Biology, Indiana University School of Medicine and the Department of Veterans Affairs, Richard L. Roudebush VAMC, Indianapolis, IN 46202, U.S.A.
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Mark A. Deeg
Mark A. Deeg
1
1Department of Medicine and Department of Biochemistry and Molecular Biology, Indiana University School of Medicine and the Department of Veterans Affairs, Richard L. Roudebush VAMC, Indianapolis, IN 46202, U.S.A.
1To whom correspondence should be addressed, at Endocrinology 111E, Indiana University, 1481 W. 10th St., Indianapolis, IN 46202, U.S.A. (email mdeeg@iupui.edu).
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Publisher: Portland Press Ltd
Received:
April 22 2005
Revision Received:
June 02 2005
Accepted:
June 08 2005
Accepted Manuscript online:
June 08 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 391 (2): 285–289.
Article history
Received:
April 22 2005
Revision Received:
June 02 2005
Accepted:
June 08 2005
Accepted Manuscript online:
June 08 2005
Citation
Nandita S. Raikwar, Rosario F. Bowen, Mark A. Deeg; Mutating His29, His125, His133 or His158 abolishes glycosylphosphatidylinositol-specific phospholipase D catalytic activity. Biochem J 15 October 2005; 391 (2): 285–289. doi: https://doi.org/10.1042/BJ20050656
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