The homotetrameric phosphoseryl-tRNA synthetase from Methanosarcina mazei exhibits half-of-the-sites activity

Abstract

Synthesis of cysteinyl-tRNA(Cys) in methanogenic archaea proceeds by a two-step pathway in which tRNA(Cys) is first aminoacylated with phosphoserine by phosphoseryl-tRNA synthetase (SepRS). Characterization of SepRS from the mesophile Methanosarcina mazei by gel filtration and nondenaturing mass spectrometry shows that the native enzyme exists as an alpha4 tetramer when expressed at high levels in Escherichia coli. However, active site titrations monitored by ATP/PP(i) burst kinetics, together with analysis of tRNA binding stoichiometry by fluorescence spectroscopy, show that the tetrameric enzyme binds two tRNAs and that only two of the four chemically equivalent subunits catalyze formation of phosphoseryl adenylate. Therefore, the phenomenon of half-of-the-sites activity, previously described for synthesis of 1 mol of tyrosyl adenylate by the dimeric class I tyrosyl-tRNA synthetase, operates as well in this homotetrameric class II tRNA synthetase. Analysis of cognate and noncognate reactions by ATP/PP(i) and aminoacylation kinetics strongly suggests that SepRS is able to discriminate against the noncognate amino acids glutamate, serine, and phosphothreonine without the need for a separate hydrolytic editing site. tRNA(Cys) binding to SepRS also enhances the capacity of the enzyme to discriminate among amino acids, indicating the existence of functional connectivity between the tRNA and amino acid binding sites of the enzyme.

FIGURE 1.

Characterization of M. mazei SepRS. A, gel filtration of SepRS (0.4 nmol of tetramer) on Superdex 200 calibrated by known molecular weight markers (see “Experimental Procedures”). B, electrospray mass spectrometry showing peaks corresponding to the α₄ SepRS enzyme. The net positive charge (z) and the precise value of m/z for each peak are indicated. The size of the tetramer is obtained by multiplying the m/z value for each peak by the corresponding charge. Each peak provides independent information confirming the size of the protein at ∼256 kDa, corresponding to a tetrameric quaternary structure.

FIGURE 2.

tRNA binding affinity and stoichiometry of SepRS. A, rapid initial depletion of ATP by SepRS, in the first step reaction forming phosphoseryl adenylate from phosphoserine and ATP, performed in the presence of inorganic pyrophosphatase. B, quenching of intrinsic tryptophan fluorescence of SepRS by tRNA binding. Shown are emission scans taken as tRNA^Cys. The concentration of tRNA was titrated from 0 (maximum emission at 340 nm) to 8 μm. The fluorescence intensity on the ordinate is given in arbitrary units. C, determination of the stoichiometry of tRNA binding to SepRS by tryptophan fluorescence quenching.

FIGURE 3.

ATP-PP_i exchange by SepRS. Thin layer chromatogram of a reaction time course showing separation of [³²P]PP_i from [³²P]ATP and [³²P]P_i. The first lane on the left is a control reaction in which SepRS was omitted.

FIGURE 4.

Steady-state aminoacylation by SepRS. A, time course for plateau phosphoserylation by SepRS in the absence or presence of ^m1G37 in tRNA^Cys. The inset shows the imaged TLC plate at the respective time points. The percentage of tRNA that is aminoacylated (plateau value) is determined directly by the ratio of intensities for Sep-AMP and AMP. B, dependence of reaction rate on amino acid concentration, used to derive k_cat and K_m for the cognate SepRS reaction. C, the active site of A. fulgidus SepRS showing amino acids that interact directly with the substrate phosphoserine (8).