Characterization of S-nitrosylation of human triose phosphate isomerase

ROMERO, JORGE M.; CARRIZO, MARÍA E.; CURTINO, JUAN A.

Rio de Janeiro

Congreso; 5th Latin American Protein Society Meeting (LAPSM); 2016

Latin American Protein Society

Nitric oxide (NO) is a diatomic gaseous free radical produced in large amounts by macrophages upon the induction of NO synthetase. One of the mayor consequences of NO exposure is the generation of the protein S-nitrosylation, which could produce alterations in protein function. Even though the S-nitrosylation mechanism in vivo is unclear, several reactive nitrogen species were proposed as mediators including NO gas (or its oxidized form N2O3), or non protein S-nitrosothiols as S-nitrosocysteine (CySNO) and S-nitrosoglutathione (GSNO). Triosephosphate isomerase (TPI) has been frequently identified as a target S-nitrosylation by proteomic studies. TPI is a glycolytic enzyme that catalyzes the isomerization of dihydroxyacetone phosphate (DHAP) to glyceraldehydes-3-phosphate (G3P), and it is found in almost all organisms. However the effects of S-nitrosylation on activity were only explored in plants and algas. Here, we show results about the S-nitrosylation of human TPI (hTPI), and the effect on its enzymatic parameters. hTPI is S-nitrosylated in time-dependent manner by releasing NO donor (Spermine NONOate), CySNO and GSNO, indicating that the mechanism involved could be mediated by both, N2O3 and transfer of NO from S-nitrosothiols to hTPI cysteine. It was determined around 0.8 mol of S-nitrosothiols/mol protein, and free thiols levels were reduced about by 15%, suggesting that just one cysteine residue was modified. Surface accessibility calculation for the five cysteines using the crystallographic coordinate of hTPI showed that Cys86 is highly accessible. hTPI S-nitrosylation produced a 25% inhibition of the Vmax of the DHAP to G3P conversion. However we did not observe effect on Km, indicating that transition state could be perturbed. This is the first study in vitro of the hTPI S-nitrosylation. Further studies will be required to identify the modified cysteine and the structural basis of inhibition.