Hemin Binds to Human Cytoplasmic Arginyl-tRNA Synthetase and Inhibits Its Catalytic Activity

Fang Yang,Xian Xia,Hui-Yan Lei,En-Duo Wang

doi:10.1074/jbc.m110.159913

Abstract

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.

Highlights

The band corresponding to ⌬NhcArgRS was observed in a gel by direct CL imaging for the protein incubated with hemin, suggesting that hemin formed a complex with the enzyme (Fig. 1C)
We investigated the effects of hemin on human cytoplasmic arginyl-tRNA synthetase (ArgRS) (hcArgRS) and found that hemin binds to the enzyme and inhibits its activity in vitro
By investigating the spectral changes of mutant proteins in the absence and presence of hemin, we identified Cys-115 as an axial ligand for hemin binding; Cys-115 is located in the unique Add1 domain of ArgRS, a domain missing in other class I aaRSs [4]

Summary

EXPERIMENTAL PROCEDURES

Materials—Hemin, hemin-agarose, L-arginine, dithiothreitol, tetrasodium pyrophosphate, ATP, Tris-HCl, magnesium chloride, sodium chloride, potassium chloride, isopropyl 1-thio-␤-D-galactopyranoside, 5,5Ј-dithiobis(2-nitrobenzoic acid) (DTNB), and activated charcoal were purchased from Sigma. Gel Filtration Chromatography—⌬NhcArgRS preincubated with hemin at a molar ratio of 1:1 was applied to a Superose-12 column for high-performance liquid chromatography (HPLC) and eluted at a flow rate of 0.5 ml/min by using a buffer containing 50 mM Tris-HCl (pH 7.5) and 100 mM NaCl. UV light-absorbing fractions that contained ⌬NhcArgRS were collected and concentrated with an Amicon Ultra-15 centrifugal filter. Assays of Aminoacylation and ATP-PPi Exchange—The aminoacylation activity of ArgRS was determined at 37 °C in a reaction mixture containing 50 mM Tris-HCl (pH 7.5), 12 mM MgCl2, 80 mM KCl, 0.1 mM EDTA, 0.05 mg/ml BSA, 20 ␮M isolated E. coli tRNAArg(ACG), 20 ␮M [3H]arginine (450 cpm/ pmol), and 3 nM ⌬NhcArgRS in the absence or presence of hemin at various concentrations. The reaction was performed in 50 mM Tris-HCl (pH 7.5), 12 mM MgCl2, 4 mM ATP, 80 mM KCl, 0.1 mM EDTA, 0.05 mg/ml BSA, 20 ␮M E. coli tRNAArg, 2 mM arginine, 2 mM tetrasodium [32P]pyrophosphate (10 cpm/ pmol), and 3 nM ⌬NhcArgRS

RESULTS

His mutants

DISCUSSION