Adenosine-5'-phosphosulfate kinase (ATP:adenylylsulfate 3'-phosphotransferase), the second enzyme in the pathway of sulfate activation, has been purified (approximately 300-fold) to homogeneity from an Escherichia coli K12 strain, which overproduces the enzyme activity (approximately 100-fold). The purified enzyme has a specific activity of 153 mumol of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) formed/min/mg of protein at 25 degrees C. The enzyme is remarkably efficient with a Vmax/Km(APS) of greater than 10(8) M-1 s-1, indicating that at physiologically low substrate concentrations the reaction is essentially diffusion limited. Upon incubation with MgATP a phosphorylated enzyme is formed; the isolated phosphorylated enzyme can transfer its phosphoryl group to adenosine 5'-phosphosulfate (APS) to form PAPS or to ADP to form ATP. The phosphorylated enzyme exists as a dimer of identical 21-kilodalton subunits, while the dephosphorylated form primarily exists as a tetramer. Divalent cations are required for activity with Mg(II), Mn(II), Co(II), and Cd(II) activating. Studies of the divalent metal-dependent stereoselectivity for the alpha- and beta-phosphorothioate derivatives of ATP indicate metal coordination to at least the alpha-phosphoryl group of the nucleotide. Steady state kinetic studies of the reverse reaction indicate a sequential mechanism, with a rapid equilibrium ordered binding of MgADP before PAPS. In the forward direction APS is a potent substrate inhibitor, competitive with ATP, complicating kinetic studies. The primary kinetic mechanism in the forward direction is sequential. Product inhibition studies at high concentrations of APS suggest an ordered kinetic mechanism with MgATP binding before APS. At submicromolar concentrations of APS, product inhibition by both MgADP and PAPS is more complex and is not consistent with a solely ordered sequential mechanism. The formation of a phosphorylated enzyme capable of transferring its phosphoryl group to APS or to MgADP suggests that a ping-pong pathway in which the rate of MgADP dissociation is comparable to the rate of APS binding might contribute at very low concentrations of APS. The substrate inhibition by APS is consistent with APS binding to the enzyme, to form a dead-end E.APS complex.