Manganese ion, like Mg2+, has been found to produce high biosynthetic activity of the unadenylylated form of glutamine synthetase obtained from Mycobacterium smegmatis, and the activity with each of these cations was decreased by the adenylylation of the enzyme. Further, the gamma-glutamyltransferase reaction was catalyzed in the presence of either Mn2+, Mg2+, or Co2+ with both unadenylylated and adenylylated enzyme; however, each of these divalent cation-dependent activities was also decreased by one order of magnitude by adenylylation of the enzyme. From studies of UV-difference spectra, it was found that the ability of M. smegmatis glutamine synthetase to assume a number of distinctly different configurations was the result of the varied response of the enzyme to different cations. When either Mn2+, Mg2+, Ca2+, or Co2+ was added to the relaxed (divalent cation-free) enzyme at saturated concentration, each produced a similar UV-difference spectrum of the enzyme, indicating that the conformational states induced by these cations are similar with respect to the polarity of the microenvironment surrounding the tyrosyl and tryptophanyl groups of the enzyme. The binding of Cd2+, Ni2+, or Zn2+ to the relaxed enzyme each produced a different shift in the UV-absorption spectrum of the enzyme, indicating different conformational states. The kinetics of the spectral change that occurred upon addition of Mn2+, Mg2+, or Co2+ to a relaxed enzyme preparation were determined. The first-order rate constants for the decrease in relaxed enzyme with Mn2+ and Mg2+ were 0.604 min-1 and 0.399 min-1, respectively, at 25 degrees C, pH 7.4. The spectral change with Co2+ was completed within the time of mixing (less than 4 s). For these three metal ions, the total spectral change as well as the time course of the change were the same for both the unadenylylated enzyme and the partially adenylylated enzyme. However, Hill coefficients obtained from spectrophotometric titration data for both Mn2+ and Mg2+ were decreased with adenylylated enzyme to compared with unadenylylated enzyme. These results suggest that covalently bound AMP on each subunit may be involved in subunit interactions within the dodecamer. Circular dichroism measurements also indicated that the various structural changes of the M. smegmatis glutamine synthetase were produced by the binding of the divalent cations.