Inactivation of rabbit muscle glycogen phosphorylase b by peroxynitrite revisited: Does the nitration of Tyr613 in the allosteric inhibition site control enzymatic function?

There is increasing evidence that sequence-specific formation of 3-nitrotyrosine (3-NT) may cause functional changes in target proteins. Recently, the nitration of Tyr residues in glycogen phosphorylase b (Phb) was implicated in the age-associated decline of protein function [Sharov et al., Exp. Gerontol. 41 (2006) 407-416]; in another report, the nitration of one specific residue, Tyr(613), located in the allosteric inhibition site was hypothesized as a rationale for perozynitrite inactivation [Dairou et al., J. Mol. Biol. 372 (2007) 1009-1021]. In this study, we have optimized the analysis of in-gel Ph-b digests by high performance liquid chromatography-electro spray ionization-tandem mass spectrometry, in order to achieve a quantitative analysis of nitration of individual Tyr residues at a high coverage of Tyr-containing sequences (92%). Our-data do not confirm the role of Tyr(613) nitration in the control of enzymatic function. Furthermore, we show here that the enzymatic activity of Ph-b does not directly correlate with the protein nitration levels, and that the modification of Cys and, potentially, other amino acid residues can better rationalize Ph-b inactivation by peroxynitrite. (C) 2008 Elsevier Inc. All rights reserved,