Impact of Substrate Protonation and Tautomerization States on Interactions with the Active Site of Arginase I

Abstract

Human arginase is a binuclear manganese metalloenzyme that participates in the urea cycle. Arginase catalyzes the hydrolysis of L-arginine into L-ornithine and urea and is linked to several disorders such as asthma and cancer. Currently, the protonation and tautomerization state of the substrate when bound to the active site, which contains two manganese ions, is not known. Knowledge of the charge-dependent behavior of arginine in the arginase I environment would be of utility toward understanding the catalytic mechanism and designing inhibitors of this enzyme. The arginine(+/0) species, including all possible neutral tautomers, were modeled using an aminoimidazole analog as template. All-atom molecular dynamics simulations were then performed on each of the charged and neutral species. In addition, a hydroxide ion was included in selected simulations to test its importance. Results show that the positively charged state of arginine is stable in the active site of arginase I, with that stabilization facilitated by the presence of hydroxide. Glu277 is indicated to play a role in stabilizing arginine in the active site and facilitating its ability to assume a catalytically competent conformation in the presence of hydroxide. The reported interactions and modeled arginine-bound arginase I structures can be used as a tool for structure-based inhibitor design, as experimental data on the structure of the substrate-enzyme complex is lacking.