Amino acid substitutions at the subunit interface of dimeric Escherichia coli alkaline phosphatase cause reduced structural stability

The consequences of amino acid substitutions at the dimer interface for the strength of the interactions between the monomers and for the catalytic fu nction of the dimeric enzyme alkaline phosphatase from Escherichia coli hav e been investigated. The altered enzymes R10A, R10K, R24A, R24K, T59A, and R10A/R24A, which have amino acid substitutions at the dimer interface, were characterized using kinetic assays, ultracentrifugation, and transverse ur ea gradient gel electrophoresis. The kinetic data for the wild-type and alt ered alkaline phosphatases show comparable catalytic behavior with k(cat) v alues between 51.3 and 69.5 s(-1) and K-m values between 14.8 and 26.3 mu M . The ultracentrifugation profiles indicate that the wild-type enzyme is mo re stable than all the interface-modified enzymes. The wild-type enzyme is dimeric in the pH range of pH 4.0 and above, and disassembled at pH 3.5 and below. All the interface-modified enzymes, however, are apparently monomer ic at pH 4.0, begin assembly at pH 5.0, and are not fully assembled into th e dimeric form until pH 6.0. The results from transverse urea gradient gel electrophoresis show clear and reproducible differences both in the positio n and the shape of the unfolding patterns; all these modified enzymes are m ore sensitive to the denaturant and begin to unfold at urea concentrations between 1.0 and 1.5 M; the wild-type enzyme remains in the folded high mobi lity form beyond 2.5 M urea. Alkaline phosphatase H370A, modified at the ac tive site and not at the dimer interface, resembles the wild-type enzyme bo th in ultracentrifugation and electrophoresis studies. The results obtained suggest that substitution of a single amino acid at the interface sacrific es not only the integrity of the assembled dimer, bur also the stability of the monomer fold, even though the activity of the enzyme at optimal pH rem ains unaffected and does not appear to depend on interface stability.