Characterization of a monomeric Escherichia coli alkaline phosphatase formed upon a single amino acid substitution.
Alkaline phosphatase (AP) from Escherichia coli as well as APs from many other organisms exist in a dimeric quaternary structure. Each monomer contains an active site located 32 A away from the active site in the second subunit. Indirect evidence has previously suggested that the monomeric form of AP is inactive. Molecular modeling studies indicated that destabilization of the dimeric interface should occur if Thr-59, located near the 2-fold axis of symmetry, were replaced by a sterically large and charged residue such as arginine. The T59R enzyme was constructed and characterized by sucrose-density gradient sedimentation, size-exclusion chromatography, and circular dichroism (CD) and compared with the previously constructed T59A enzyme. The T59A enzyme was found to exist as a dimer, whereas the T59R enzyme was found to exist as a monomer. The T59A, T59R, and wild-type APs exhibited almost identical secondary structures as judged by CD. The T59R monomeric AP has a melting temperature (Tm) of 43 degrees C, whereas the wild-type AP dimer has a Tm of 97 degrees C. The catalytic activity of the T59R enzyme was reduced by 104-fold, whereas the T59A enzyme exhibited an activity similar to that of the wild-type enzyme. The T59A and wild-type enzymes contained similar levels of zinc and magnesium, whereas the T59R enzyme has almost undetectable amounts of tightly bound metals. These results suggest that a significant conformational change occurs upon dimerization, which enhances thermal stability, metal binding, and catalysis.
Boulanger
Robert R
RR
Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, USA.
Kantrowitz
Evan R
ER
D000469:Alkaline Phosphatase / Q000737:chemistry*; D019943:Amino Acid Substitution; D002499:Centrifugation, Density Gradient; D002942:Circular Dichroism; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006358:Hot Temperature; D007700:Kinetics; D008274:Magnesium / Q000032:analysis; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D015032:Zinc / Q000032:analysis
D029968:Escherichia coli Proteins; D021122:Protein Subunits; D000469:Alkaline Phosphatase; D008274:Magnesium; D015032:Zinc