Fructose-1,6-bisphosphatase requires a divalent metal cation for catalysis, Mg(2+) being its most studied activator. Phosphatase activity increases sigmoidally with the concentration of Mg(2+), but the mechanistic basis for such cooperativity is unknown. Bound magnesium cations can interact within a single subunit or between different subunits of the enzyme tetramer. Mutations of Asp(118), Asp(121), or Glu(97) to alanine inactivate the recombinant porcine enzyme. These residues bind directly to magnesium cations at the active site. Three different hybrid tetramers of fructose-1,6-bisphosphatase, composed of one wild-type subunit and three subunits bearing one of the mutations above, exhibit kinetic parameters (K(m) for fructose-1,6-bisphosphate, 1.1-1.8 microm; K(a) for Mg(2+), 0.34-0.76 mm; K(i) for fructose-2,6-bisphosphate, 0.11-0.61 microm; and IC(50) for AMP, 3.8-7.4 microm) nearly identical to those of the wild-type enzyme. Notwithstanding these similarities, the k(cat) parameter for each hybrid tetramer is approximately one-fourth of that for the wild-type enzyme. Evidently, each subunit in the wild-type tetramer can independently achieve maximum velocity when activated by Mg(2+). Moreover, the activities of the three hybrid tetramers vary sigmoidally with the concentration of Mg(2+) (Hill coefficients of approximately 2). The findings above are fully consistent with a mechanism of cooperativity that arises from within a single subunit of fructose-1,6-bisphosphatase.