Hemin binds to human cytoplasmic arginyl-tRNA synthetase and inhibits its catalytic activity

The research team led by Prof. Enduo Wang (SIBCB) recently discovered that hemin binds to human cytoplasmic arginyl-tRNA synthetase and inhibits its catalytic activity. The result was published in J. Biol. Chem. on October 5th.

 

The free form of human cytoplasmic arginyl-tRNA synthetase (hcArgRS) is hypothesized to participate in ubiquitin-dependent protein degradation by offering arginyl-tRNAArg to arginyl-tRNA transferase (ATE1). In the study, the researchers investigated the effect of hemin on hcArgRS based on the fact that hemin regulates several critical proteins in "N-end rule"protein degradation pathway. Extensive biochemical evidence has established that hemin could bind to both forms of hcArgRS in vitro. Based on the spectral changes of the Soret band on site-directed protein mutants, the researchers identified Cys-115 as a specific axial ligand of hemin binding that is located in the Add1 domain. Hemin inhibited the catalytic activity of full-length and N-terminal 72-amino acid truncated hcArgRSs by blocking amino acid activation. Kinetic analysis demonstrated that the Km values for tRNAArg, arginine and ATP in the presence of hemin were not altered, but kcat values dramatically decreased compared with those in the absence of hemin. By comparison, the activity of prokaryotic ArgRS was not affected obviously by hemin. Gel filtration chromatography suggested that hemin induced oligomerization of both the isolated Add1 domain and the wild type enzyme, which could account for the inhibition of catalytic activity. However, the catalytic activity of a hcArgRS mutant with Cys-115 replaced by alanine (hcArgRS-C115A) was also inhibited by hemin, suggesting that hemin binding to Cys-115 is not responsible for the inhibition of enzymatic activity and that the specific binding may participate in other biological functions. Prof. Wang’s result provides a valuable clue to further investigate the new biological function of mammals ArgR.