tailieunhanh - Báo cáo khoa học:Site-directed mutagenesis of mouse glutathione transferase P1-1 unlocks masked cooperativity, introduces a novel mechanism for ‘ping pong’ kinetic behaviour, and provides further structural evidence for participation of a water molecule in proton abstraction from glutathione

Mouse liver glutathione transferase P1-1 has three cysteine residues at posi-tions 14, 47 and 169. We have constructed the single, double and triple cys-teine to alanine mutants to define the behaviour of all three thiols. We confirm that C47 is the ‘fast’ thiol (), and define C169 as the alkaline reactive residue with a pKa of . | 1FEBS Journal Site-directed mutagenesis of mouse glutathione transferase P1-1 unlocks masked cooperativity introduces a novel mechanism for ping pong kinetic behaviour and provides further structural evidence for participation of a water molecule in proton abstraction from glutathione Gavin McManus1 Marta Costa2 Albert Canals2 3 Miquel Coll2 3 and Timothy J. Mantle1 1 Schoolof Biochemistry and Immunology Trinity College Dublin 2 Ireland 2 Institut de Biologia Molecular de Barcelona CSIC Barcelona Science Park Barcelona Spain 3 Institute for Research in Biomedicine Barcelona Science Park Barcelona Spain Keywords cooperativity enzyme mechanism GST kinetics thiolreactivity Correspondence G. McManus Schoolof Biochemistry and Immunology Trinity College Dublin 2 Ireland Fax 00 35 31 6772400 Tel 00 35 31 8961612 E-mail gjmcmanus@ Received 21 September 2010 revised 26 October 2010 accepted 2 November 2010 doi Mouse liver glutathione transferase P1-1 has three cysteine residues at positions 14 47 and 169. We have constructed the single double and triple cysteine to alanine mutants to define the behaviour of all three thiols. We confirm that C47 is the fast thiol pK and define C169 as the alkaline reactive residue with a pKa of . Only a small proportion of C14 is reactive with 5 5 -dithiobis- 2-nitrobenoic acid DTNB at pH 9 in the C47A C169A double mutant. The native enzyme and the C169A mutant exhibited Michaelis-Menten kinetics but all other thiol to alanine mutants exhibited sigmoidal kinetics to varying degrees. The C169A mutant exhibited ping pong kinetics consistent with a mechanism whereby liberation of a proton from a reduced enzyme-glutathione GSH complex to form an enzymeGS unprotonated complex is essentially irreversible. Intriguingly similar behaviour has recently been reported for a mutant of the yeast prion Ure2p. This cooperative behaviour is mirrored in the crystal structure of the C47A mutant which binds the .

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