Architectural underpinnings of the genetic code for glutamine.

Structure-based mutational analysis was used to probe the architecture of the glutamine binding pocket in Escherichia coli glutaminyl-tRNA synthetase (GlnRS). Crystallographic studies of several different GlnRS complexes in a lattice that supports catalytic activity have shown that the glutamine amide group makes only ambiguous hydrogen-bonding interactions with a tyrosine hydroxyl and bound water molecule, rather than the highly specific hydrogen-bonding and electrostatic interactions made by the substrate amino acid in all other nonediting tRNA synthetases. Further, the amide oxygen of substrate glutamine accepts a hydrogen bond from the 3'-ribose hydroxyl group of ATP, an unusual distal substrate-substrate interaction also not observed in any other tRNA synthetase complex. Steady-state and pre-steady-state kinetic analysis using a 3'-dATP analogue in place of ATP shows that removal of this distal interaction does not affect K(m) for the analogue as compared with ATP, yet decreases the efficiency of aminoacylation by 10(3)-fold while significantly elevating K(m) for glutamine. In other experiments, mutation of eight nearly fully conserved residues in the glutamine binding pocket reveals decreases in k(cat)/K(m) ranging from 5- to 400-fold, and in K(d) for glutamine of up to at least 60-fold. Amino acid replacements at Tyr211 and Gln255, which participate with substrate glutamine in an antidromic circular arrangement of hydrogen bonds, cause the most severe decreases in catalytic efficiency. This finding suggests that the relative absence of direct hydrogen bonds to glutamine may be in part compensated by additional binding energy derived from the enhanced stability of this circular network. Calculations of electrostatic surface potential in the active site further suggest that a complementary electrostatic environment is also an important determinant of glutamine binding.

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