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

Bromomethyl ketone derivatives of L-valine (VBMK), L-isoleucine (IBMK), L-n orleucine (NleBMK) and L-phenylalanine (FBMK) were synthesized. These reage nts were used for qualitative comparative labeling of Escherichia coli valy L-tRNA synthetase (ValRS), an enzyme with Val/Ile editing activity, in orde r to identify the binding sites for L-valine or noncognate amino acids. Lab eling of E. coli ValRS with the substrate analog valyL-bromomethyl ketone ( VBMK) resulted in a complete loss of valine-dependent isotopic [P-32]PPi-AT P exchange activity. L-Valine protected the enzyme against inactivation. No ncognate amino acids analogs isoleucyL-, norleucyL- and phenylalanyL-bromom ethyl ketones (IBMK, NleBMK and FBMK) were also capable of abolishing the a ctivity of ValRS, FBMK being less efficient in inactivating the synthetase. Matrix-assisted laser desorption-ionization mass spectrometry designated c ysteines 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 ke tones. With the exception of Cys829, which was most likely unspecifically l abeled, the amino-acid residues labeled by the reagents derived from noncog nate 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 labele d in common by all the used bromomethyl ketone derivatives of noncognate am ino acids, suggesting that the synthetic site where aminoacyl adenylate for mation takes place on E. coli ValRS is built up of two subsites. One subsit e containing Cys424 might represent the catalytic locus of the active cente r where specific L-valine activation takes place. The second subsite contai ning His433 might represent the binding site for noncognate amino acids. Th e fact that Cys275 and His282, fragment 259-291, were labeled by IBMK, NleB MK and FBMK, but not by the substrate analog VBMK, suggests that these resi dues might be located at or near the editing site of E. coli ValRS. Compari son of fragment 259-291 with all the available ValRS amino-acid sequences r evealed that His282 is strictly conserved, with the exception of its replac ement by a glycine in a subgroup corresponding to the archaebacteria. Becau se 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 propose d that the conserved His282 of E. coli ValRS is involved in editing.