Characterization of a Monomeric Escherichia coli Alkaline Phosphatase Formed upon a Single Amino Acid Substitution

Abstract

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 Å 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 (T m ) of 43°C, whereas the wild-type AP dimer has a T m of 97°C. The catalytic activity of the T59R enzyme was reduced by 10 4 -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.In some oligomeric proteins, the isolated monomeric subunits must be assembled before the protein becomes functional. Such behavior is exemplified by studies on homodimeric chorismate mutase (1) and the trimeric catalytic subunit of aspartate transcarbamoylase (2). In both of these enzymes, the active site contains residues donated from neighboring chains, and each active site is located at the interface between subunits. The reasons for obligatory subunit association are less obvious for enzymes that do not have a shared active site or exhibit allosteric control and regulation. For example, triosephosphate isomerase (3, 4) and dihydroorotate dehydrogenase A (5) are both dimeric enzymes that have active sites in which all the catalytically important residues are derived from a single polypeptide chain. Nevertheless, monomeric mutants of triose-phosphate isomerase and dihydroorotate dehydrogenase A were found to lack structural stability and to exhibit the absence or reduction of activity. A monomeric form of a dimeric enzyme that contains an unshared active site in each subunit, Escherichia coli alkaline phosphatase (phosphomonoester hydrolase (EC 3.1.3.1)), is characterized in this study.Alkaline phosphatase is a homodimeric metalloenzyme, which hydrolyzes phosphomonoesters into inorganic phosphate and the corresponding alcohol. The dimer contains two active sites, located 32 Å apart, each of which accommodates a magnesium and two zinc ions (6). It has been determined that all three metals play a role in the...

Engineered Monomeric Human Histidine Triad Nucleotide-binding Protein 1 Hydrolyzes Fluorogenic Acyl-adenylate and Lysyl-tRNA Synthetase-generated Lysyl-adenylate

“…1D). The substitution of charged residues, such as Asp, Arg, and Glu into a protein dimer or oligomer interface has been shown to be a viable approach for preparing monomeric enzymes (25,(37)(38)(39).…”

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Molecular Dissection of Erv26p Identifies Separable Cargo Binding and Coat Protein Sorting Activities

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Characterization of a monomeric heat-labile classical alkaline phosphatase from Anabaena sp. PCC7120

“…Previous work on EAP showed that the introduction of amino acids with large, bulky, and charged side chains into the interface of EAP disturbs the formation of the dimeric structure. The replacement of Thr59 with Arg resulted in the formation of a monomeric EAP mutant with little activity, and in B. subtilis APIV, a monomeric PhoA, the corresponding residue is also Arg [53]. Amino acid align ments showed that the residue in AP A , which corresponds to Thr59 in EAP, is also an Arg.…”