Structure/function studies and protein engineering of ATP-dependent peptide ligases.

Glutathione is found in mammals, plants, and bacteria and is synthesized by glutathione synthetase (GS). Some plants synthesize glutathione analogs in which ?-alanine replaces glycine. The chemical diversity of these molecules suggests that the substrate specificity of the GS-like peptide ligases differs from GS, but the molecular basis for this variety remains unclear. To better understand the reaction mechanism of Arabidopsis GS (AtGS) site-directed mutagenesis was performed. Residues interacting with bound magnesium ions (E148, N150, E371, and E429) were essential for activity. K313 and K367 were important for ATP binding. Kinetic analysis suggests that R132 provides a key catalytic role. To define the determinants of substrate specificity, we compared AtGS (glycine specific) with Glycine max (soybean) homoglutathione synthetase (GmhGS; ?-alanine specific). Sequence alignments suggest that two active site residues (AtGS: A466 and A467; GmhGS: L466 and P467) modulate substrate preference between glycine and ?-alanine. The interconversion of AtGS to GmhGS and vice versa is described. GmhGS displays a 700-fold preference for ?-alanine, whereas, the double mutant accepts either substrate with equal efficiency. This suggests that differences outside the active site further define substrate specificity in this family of enzymes, and emphasize the need for crystallographic studies. [ABSTRACT FROM AUTHOR]