The free form of human cytoplasmic arginyl-tRNA synthetase (hcArgRS) is hypothesized to participate in ubiquitin-dependent protein degradation by offering arginyl-tRNA Arg to arginyl-tRNA transferase (ATE1). We investigated the effect of hemin on hcArgRS based on the fact that hemin regulates several critical proteins in the "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, we 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 K m values for tRNA Arg , arginine, and ATP in the presence of hemin were not altered, but k cat 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 an 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.Aminoacyl-tRNA synthetase (aaRS) 2 plays an essential role in the first step of protein biosynthesis by catalyzing the attachment of amino acids to the 3Ј-end of their cognate tRNAs. Based on the architecture of their catalytic sites, the 20 aaRSs can be classified into two groups (1). Class I aaRSs have "HIGH" and "KMSKS" signature sequences located in the Rossmann fold active site. Class II aaRSs are characterized by three homologous motifs (1). Usually, the reaction catalyzed by aaRSs occurs in two steps: amino acid activation followed by aminoacylation of the cognate tRNA. The amino acid activation of aaRSs does not require the presence of the cognate tRNA except for arginyl-tRNA synthetase (ArgRS), glutamyl-tRNA synthetase (GluRS), and glutaminyl-tRNA synthetase (GlnRS), which activate their cognate amino acids in a tRNA-dependent manner (2, 3).ArgRS is a class I aaRS, and tRNA binding is a prerequisite for activating arginine. The crystal structures of ArgRS from several species have been solved (4 -6). Saccharomyces cerevisiae cytoplasmic ArgRS (yArgRS) is a monomeric protein with five domains: the N-terminal additional domain (Add1), the catalytic domain including two insertion domains (Ins-1 and Ins-2), and the C-terminal additional domain (Add2). The tRNA Arg serves as the activator of ArgRS in the reaction of amino acid activation and as the substrate in aminoacylation. Three regions of ArgRS are involved in recognizing tRNA Arg : the ac...