Temperature-Sensitive Mutant of Bacillus subtilis Glutamine Synthetase Obtained by Random Mutation

Abstract

Random mutations were introduced into the B. subtilis glutamine synthetase gene by using nitrous acid, and a high temperature-sensitive mutant was selected. DNA sequencing of the restriction fragment containing the mutation revealed a single base-pair change resulting in the substitution of Leu 318 with Phe. The mutant enzyme was purified, and its kinetic and physical properties were characterized. The Mg²⁺-dependent activity and Mg²⁺ plus Mn²⁺-dependent activity of the mutant were less than 5% of those of the wild-type at 37°C, and these activities decreased above 15°C, whereas the Mn²⁺ dependent activity was nearly normal. Affinity of the mutant enzyme for glutamate was extremely decreased although the K_m values for NH₃ or ATP were almost the same as those of the wild-type. The mutant enzyme was more susceptible than the wild-type enzyme to digestion with chymotrypsin in the presence of glutamate, ATP, and Mg²⁺, although addition of glutamate, ATP, and Mn²⁺ completely protected both enzymes. These results and circular dichroism analyses suggested that Leu 318 is at the glutamate-binding site and that the substitution of Leu 318 for Phe reduces the ability of the enzyme to form the enzyme-substrate complex, probably supported by Mg²⁺.