Characterization of heterodimeric alkaline phosphatases from Escherichia coli: An investigation of intragenic complementation

Escherichia coli alkaline phosphatase (EC 3.1.3.1) belongs to a rare group of enzymes that exhibit intragenic complementation. When certain mutant ver sions of alkaline phosphatase are combined, the resulting heterodimeric enz ymes exhibit a higher level of activity than would be expected based upon t he relative activities of the parental enzymes. Nine previously identified alkaline phosphatase complementation mutants were re-examined in this work in order to determine a molecular explanation of intragenic complementation in this experimental system. The locations of these mutations were determi ned by DNA sequence analysis after PCR amplification of the phosphatase-neg ative phoA gene. Most of the mutations involved ligands to metal-binding si tes. Each of the mutant enzymes was re-created by site-specific mutagenesis , expressed, purified, and kinetically characterized. To investigate cooper ativity between the two subunits, we analyzed heterodimeric forms of some o f the site-specific mutant enzymes. To enable the isolation of the heterodi meric alkaline phosphatase in pure form, the overall charge of one subunit was altered by replacing the C-terminal Lys residue with three Asp residues . This modification had no effect on the kinetic properties of the enzyme. Heterodimeric alkaline phosphatases were created using two methods: (1) in vitro formation by dissociation at acid pH followed by reassociation at sli ghtly alkaline pH conditions in the presence of zinc and magnesium ions; an d (2) in vivo expression from a plasmid carrying two different phoA genes. Increases in k(cat), as well as a large reduction in the p-nitrophenyl phos phate K-m were observed for certain combinations of mutant enzymes. These r esults suggest that the structural assembly of E. coli alkaline phosphatase into the dimer induces cooperative interactions between the monomers neces sary for the formation of the functional form of the holoenzyme. (C) 2000 A cademic Press.