The loop from Ile-226 to Arg-241 in the glutathione synthetase (GSHase) from Escherichia coli B is rich in glycine and alanine and too flexible to take a fixed conformation [Yamaguchi, H., Kato, H., Hata, Y., Nishioka, T., Kimura, A., Oda, J., & Katsube, Y. (1993) J. Mol. Biol. 229, 1083-1100; Tanaka, T., Kato, H., Nishioka, T., & Oda, J. (1992) Biochemistry 31, 2259-2265]. To restrict the flexibility, three residues in the loop, Pro-227, Gly-229, and Gly-240, were replaced with alanine and valine residues. Variability in conformations of the mutant loops and shifts in the distribution of conformers between the open and closed states were assessed by steady-state kinetics, X-ray crystallographic structure analysis, and proteolysis with arginyl endopeptidase. Mutant enzymes replaced with a valine residue at the basal positions of the loop (P227V, G240V, and P227V/G240V) were identical with the wild-type enzyme in their crystal structures, except the loop region. The mutant loops retained apparent conformational variability, so as to take the open and closed states and to protect the acyl phosphate intermediate from the decomposition uncoupled from glutathione synthesis, but lost the catalytic activity; K(m)app values for glycine and gamma-Glu-Cys were sensitive to the mutations and drastically increased, and the k0app value was fatally reduced in the P227V/G240V mutant enzyme. The present results suggest that adjustability of the loop to the closed state is required for the recognition of the substrates, gamma-Glu-Cys and glycine, and for the chemical interactions with the bound substrates.