title: Valyl-tRNA synthetase from Escherichia coli MALDI-MS identification of the binding sites for L-valine or for noncognate amino acids upon qualitative comparative labeling with reactive amino-acid analogs.
abstract: Bromomethyl ketone derivatives of L-valine (VBMK), L-isoleucine (IBMK), L-norleucine (NleBMK) and L-phenylalanine (FBMK) were synthesized. These reagents were used for qualitative comparative labeling of Escherichia coli valyl-tRNA synthetase (ValRS), an enzyme with Val/Ile editing activity, in order to identify the binding sites for L-valine or noncognate amino acids. Labeling of E. coli ValRS with the substrate analog valyl-bromomethyl ketone (VBMK) resulted in a complete loss of valine-dependent isotopic [32P]PPi-ATP exchange activity. L-Valine protected the enzyme against inactivation. Noncognate amino acids analogs isoleucyl-, norleucyl- and phenylalanyl-bromomethyl ketones (IBMK, NleBMK and FBMK) were also capable of abolishing the activity of ValRS, FBMK being less efficient in inactivating the synthetase. Matrix-assisted laser desorption-ionization mass spectrometry designated cysteines 424 and 829 as the target residues of the substrate analog VBMK on E. coli ValRS, whereas, altogether, IBMK, NleBMK and FBMK labeled His266, Cys275, His282, His433 and Cys829, of which Cys275, His282 and His433 were labeled in common by all three noncognate amino-acid-derived bromomethyl ketones. With the exception of Cys829, which was most likely unspecifically labeled, the amino-acid residues labeled by the reagents derived from noncognate amino acids were distributed between two fragments 259-291 and 419-434 in the primary structure of E. coli ValRS. In fragment 419-434, Cys424 was specifically labeled by the substrate analog VBMK, while His433 was labeled in common by all the used bromomethyl ketone derivatives of noncognate amino acids, suggesting that the synthetic site where aminoacyl adenylate formation takes place on E. coli ValRS is built up of two subsites. One subsite containing Cys424 might represent the catalytic locus of the active center where specific L-valine activation takes place. The second subsite containing His433 might represent the binding site for noncognate amino acids. The fact that Cys275 and His282, fragment 259-291, were labeled by IBMK, NleBMK and FBMK, but not by the substrate analog VBMK, suggests that these residues might be located at or near the editing site of E. coli ValRS. Comparison of fragment 259-291 with all the available ValRS amino-acid sequences revealed that His282 is strictly conserved, with the exception of its replacement by a glycine in a subgroup corresponding to the archaebacteria. Because a nucleophile is needed in the editing site to achieve hydrolysis of an undesired product at the level of the carbonyl group thereof, it is proposed that the conserved His282 of E. coli ValRS is involved in editing.
authors.affiliation: Laboratoire de Biochimie (CNRS UMR 7654), Ecole Polytechnique, Palaiseau, France. codjo@coli.polytechnique.fr
authors.forename: C
authors.initials: C
authors.lastname: Hountondji
authors.forename: C
authors.initials: C
authors.lastname: Beauvallet
authors.forename: P
authors.initials: P
authors.lastname: Dessen
authors.forename: C
authors.initials: C
authors.lastname: Hoang-Naudin
authors.forename: J M
authors.initials: JM
authors.lastname: Schmitter
authors.forename: J C
authors.initials: JC
authors.lastname: Pernollet
authors.forename: S
authors.initials: S
authors.lastname: Blanquet
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D005033:Ethylmaleimide / Q000737:chemistry; D007532:Isoleucine / Q000378:metabolism; D007659:Ketones / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009646:Norleucine / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010649:Phenylalanine / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D016963:Thermus thermophilus / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors; D014633:Valine / Q000378:metabolism*; D014637:Valine-tRNA Ligase / Q000737:chemistry*
chemical_list: D007659:Ketones; D010455:Peptides; D007532:Isoleucine; D010649:Phenylalanine; D009646:Norleucine; D014637:Valine-tRNA Ligase; D014633:Valine; D004047:Diethyl Pyrocarbonate; D005033:Ethylmaleimide