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 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 (Tm) of 43 °C, whereas the wild-type AP dimer has a Tm of 97 °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. 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 (Tm) of 43 °C, whereas the wild-type AP dimer has a Tm of 97 °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. 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 (1MacBeath G. Kast P. Hilvert D. Science. 1998; 279: 1958-1961Google Scholar) and the trimeric catalytic subunit of aspartate transcarbamoylase (2Wente S.R. Schachman H.K. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 31-35Google Scholar). 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 (3Borchert T.V. Abagyan R. Jaenicke R. Wierenga R.K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1515-1518Google Scholar, 4Schliebs W. Thanki N. Jaenicke R. Wierenga R.K. Biochemistry. 1997; 36: 9655-9662Google Scholar) and dihydroorotate dehydrogenase A (5Ottosen M.B. Bjornberg O. Norager S. Larsen S. Palfey B.A. Jensen K.F. Protein Sci. 2002; 11: 2575-2583Google Scholar) 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 (6Sowadski J.M. Handschumacher M.D. Murthy H.M.K. Foster B.A. Wyckoff H.W. J. Mol. Biol. 1985; 186: 417-433Google Scholar). It has been determined that all three metals play a role in the catalytic mechanism of the enzyme (7Stec B. Holtz K.M. Kantrowitz E.R. J. Mol. Biol. 2000; 299: 1303-1311Google Scholar). Dimerization of the AP 1The abbreviations used are: AP, alkaline phosphatase; CD, circular dichroism. monomer occurs spontaneously upon the addition of metals, whereas AP will not dimerize in the absence of metals (8Schlesinger M.J. Barrett K. J. Biol. Chem. 1965; 240: 4284-4292Google Scholar, 9Falk M.C. Bethune J.L. Vallee B.L. Biochemistry. 1982; 21: 1471-1478Google Scholar). The two monomers of AP are held together by an extensive network of hydrophobic and hydrogen-bonding interactions along the interface. The interface of each subunit is composed of 86 amino acids, encompasses 20% of the protein, 2900 Å2 of surface area, and is comprised of a secondary structure of four α-helices and three β-strands (7Stec B. Holtz K.M. Kantrowitz E.R. J. Mol. Biol. 2000; 299: 1303-1311Google Scholar). Analysis of the interface using the most recent high resolution (1.75 Å) structure of AP as the basis (7Stec B. Holtz K.M. Kantrowitz E.R. J. Mol. Biol. 2000; 299: 1303-1311Google Scholar) reveals that there are 26 hydrogen-bonding interactions occurring among the residues of the interface. It was suggested in 1965 that these interface interactions of the AP dimer alter the tertiary structure of each subunit upon dimer formation in a manner that allows the metals to bind, creating the functional active site (10Schlesinger M.J. J. Biol. Chem. 1965; 240: 4293-4298Google Scholar). Monomers of the wild-type enzyme have been obtained through a variety of techniques such as thiol reduction in the presence of urea (11Levinthal C. Signer E.R. Fetherolf K. Proc. Natl. Acad. Sci. U. S. A. 1962; 48: 1230-1237Google Scholar), acid treatment (12Schlesinger M.J. Levinthal C. J. Mol. Biol. 1963; 7: 1-12Google Scholar), metal chelation, followed by incubation in 5% formamide (9Falk M.C. Bethune J.L. Vallee B.L. Biochemistry. 1982; 21: 1471-1478Google Scholar), or by isolation of matrix bound monomers (13McCracken S. Meighen E. J. Biol. Chem. 1980; 255: 2396-2404Google Scholar). However, the characterization of these monomeric forms of E. coli AP have been conducted under very specialized conditions. Therefore, to understand better the functional role of the dimerization process in E. coli AP, a monomeric version of the enzyme was rationally designed by altering a single amino acid side chain in the monomer-monomer interface. A stable monomeric version of the protein allowed a more direct comparison between the properties of the monomer and those of the wild-type dimeric form under benign buffer conditions. Materials—Agar, agarose, ampicillin, p-nitrophenyl phosphate, magnesium chloride, and zinc sulfate were purchased from Sigma. Tris, sucrose, and enzyme-grade ammonium sulfate were supplied by ICN Biomedicals (Costa Mesa, CA). Tryptone and yeast extract were obtained from Difco Laboratories (Detroit, MI). DNA sequencing was carried out by Beth Israel Deaconess Medical Center, Molecular Medicine Unit. The oligonucleotides required for site-specific mutagenesis and sequencing were obtained from Operon Technologies (Alameda, CA). Plasmids were isolated and purified using the QIAprep Spin Miniprep kit purchased from Qiagen (Valencia, CA). Strains and Plasmids—E. coli K12 strain MV1190 (Δ(lac-proAB), supE, thi, Δ(sri-recA) 306::Tn10(tetr)/F′ traD36, proAB, lacIq, lacZΔM15) was obtained from J. Messing. E. coli K12 strain SM547 (Δ(phoA-phoC), phoR, tsx::Tn5, Δlac, galK, galU, leu, strr) was a gift from H. Inouye. The mutations were constructed in plasmid pEK154, which contains the wild-type phoA gene and its natural promoter in pUC119. This plasmid was derived from plasmid pEK48 (14Chaidaroglou A. Brezinski J.D. Middleton S.A. Kantrowitz E.R. Biochemistry. 1988; 27: 8338-8343Google Scholar) by removal of an 882-base pair BstEII-XhoI fragment followed by treatment with T4 DNA ligase after the sticky ends had been filled out with the Klenow fragment of DNA polymerase. Plasmid pEK381 has the T59A mutation in the phoA gene introduced into pEK154 (15Martin D.C. Pastra-Landis S.C. Kantrowitz E.R. Protein Sci. 1999; 8: 1152-1159Google Scholar). Construction of a Plasmid for the Expression of the T59R Alkaline Phosphatase—The T59R mutation was induced in the phoA gene contained in the plasmid pEK154 using the procedures outlined by Stratagene in the QuikChange mutagenesis kit protocol. The entire gene was sequenced to ensure that the correct mutation was present and to confirm that no other mutations had been introduced. The final plasmid containing the T59R mutation in the phoA gene was designated pEK618. Modeling Studies—All molecular modeling studies were carried out using the program QUANTA, and energy minimization was accomplished using X-PLOR (16Brünger A.T. X-PLOR Version 3.1: a System for X-ray Crystallography and NMR. Yale University Press, New Haven, CT1992Google Scholar). The 1.75-Å wild-type AP Protein Data Bank structure 1ED8 (7Stec B. Holtz K.M. Kantrowitz E.R. J. Mol. Biol. 2000; 299: 1303-1311Google Scholar) was used as the base structure. Protein Expression—SM547, an E. coli strain with the wild-type phoA gene deleted from the chromosome and a mutation in the phoR regulatory gene, was used as the host strain for expression of the wild-type and mutant AP. The deletion of the phoA gene ensures that no alkaline phosphatase can be produced in the absence of a phoA-containing plasmid present in the host strain. Protein Purification—The wild-type, T59R, and T59A enzymes were isolated from the periplasmic space by osmotic shock and ammonium sulfate precipitation by the methods previously described (14Chaidaroglou A. Brezinski J.D. Middleton S.A. Kantrowitz E.R. Biochemistry. 1988; 27: 8338-8343Google Scholar). Final purification was carried out on a Bio-Rad BioLogic HR system using a Q-Sepharose Fast Flow column (1.6 × 10 cm). A 200-ml gradient from 0 to 0.1 m NaCl in TMZP buffer (0.01 m Tris-HCl, 0.001 m MgCl2, 10–5m ZnSO4, 10–4m NaH2PO4, 0.31 × 10–2m NaN3, pH 7.4) was used to elute the protein. The purity of the enzyme was determined by SDS-PAGE (17Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar). Determination of the Protein Concentration—The concentration of the wild-type enzyme was determined by absorbance measurements at 278 nm with an extinction coefficient of 0.71 cm2/mg (18Plocke D.J. Vallee B.L. Biochemistry. 1962; 1: 1039-1043Google Scholar). The concentration of the mutant enzymes was determined by the Bio-Rad version of Bradford's dye binding assay (19Bradford M.M. Anal. Biochem. 1976; 72: 248-254Google Scholar) using wild-type alkaline phosphatase as the standard. Determination of the Enzymatic Activity—Alkaline phosphatase activity was measured spectrophotometrically utilizing p-nitrophenyl phosphate as the substrate at 25 °C by monitoring the release of p-nitrophenolate at 410 nm (20Garen A. Levinthal C. Biochim. Biophys. Acta. 1960; 38: 470-483Google Scholar) in 1 m Tris buffer, pH 8.0. Sucrose-density Gradient Centrifugation—Sedimentation coefficients of the mutant enzymes were determined by sucrose-density gradient centrifugation. A 4.6-ml gradient of 6–25% sucrose in 50 mm NaH2PO4, pH 8.0, was of protein was on of the The were at × for using a in a The were using a A sucrose, was from the to a in to in The in to the was used to the of the gradient was accomplished using and wild-type AP as was carried out on a Bio-Rad BioLogic HR system using a Bio-Rad The column was with mm buffer, pH 8.0, at a of and using as and which have of and circular dichroism was used to from to nm and Tm for the wild-type and mutant alkaline were carried out using of enzyme in 10 mm buffer, pH 8.0. For the Tm the at nm was as the temperature in 1 °C from 25 to °C. The was allowed to at each temperature for 1 before the was Determination of was used to the metal of the the enzyme were 10 mm Tris buffer, pH 8.0, at °C for of were into the at a of 1 and compared with magnesium and zinc sulfate of a Alkaline Phosphatase—The most mutation for destabilization of dimeric AP was determined through molecular modeling a to mutation was introduced at in the AP structure Data Bank using the program and using large of the protein the of the amino acid were required to the that the mutation with dimer of the T59A and T59R Alkaline quaternary structure of the T59A and T59R enzymes was determined by both sucrose-density gradient and size-exclusion The of the wild-type and T59A enzymes are identical that have the dimeric quaternary structure. The of the T59R enzyme exhibited no as a a was The coefficients of the T59A and T59R enzymes were and upon these can be that the T59A enzyme as a dimer and the T59R enzyme as a monomer. was used to the of the T59R, T59A, and wild-type The for the T59A and wild-type enzymes were very similar with identical However, the T59R enzyme with a These results the of a dimeric structure for the T59A enzyme and a monomeric structure for the T59R enzyme. upon these the of the wild-type, the T59A, and the T59R were determined to be and of molecular can be determined from the coefficient and J. Biol. Chem. 1997; Scholar). The molecular for both the wild-type and T59A enzymes was whereas the T59R had a molecular of that the T59R enzyme is of the T59R Alkaline The T59R AP had reduced activity compared with the wild-type enzyme. the activity of the T59R AP was that of the wild-type was that for the out the that the activity was to the formation of amounts of the more active dimeric the of protein concentration on the activity were more a of protein concentration to the activity was found to be The lack of a concentration of the activity is of the absence of occurring for the T59R enzyme. in the for the wild-type and T59A enzymes are However, the of the T59R enzyme is reduced by more as compared with the wild-type for the wild-type and enzymes at pH are from the using a dimer molecular of for and T59A and a monomer molecular of for × for the T59A enzyme were previously × × The are from the using a dimer molecular of K. Proc. Natl. Acad. Sci. U. S. A. Scholar) for and T59A and a monomer molecular of for The for the T59A enzyme were previously (15Martin D.C. Pastra-Landis S.C. Kantrowitz E.R. Protein Sci. 1999; 8: 1152-1159Google Scholar). in a of on the of T59R Alkaline of measurements were to amounts of the properties of the T59R AP. the activity of the T59R AP was determined in the presence of at to used for the wild-type enzyme. In the second of the T59R AP was with 10 mm concentration at 25 °C and °C for before the activity measurements were The T59R AP in TMZP and in m Tris, pH 8.0, buffer was with followed by activity measurements in 1 m Tris, pH 8.0, with phosphate as the The activity of the T59R AP carried out in the presence of and and after incubation with 10 mm very change in activity compared with the activity of the T59R AP. Analysis of the and APs by in the of the T59R, T59A, and wild-type enzymes are of these G. R. Jaenicke R. Protein Scholar) that the three enzymes have almost identical secondary Each enzyme contained almost identical of and was used to the thermal stability of the wild-type and mutant enzymes by monitoring the change in at The Tm for the T59A, T59R, and wild-type enzymes are in The T59A enzyme a Tm of °C, °C that of the wild-type enzyme. However, the T59R mutation induced a on thermal The Tm for the T59R enzyme was 43 °C, the 97 °C for the wild-type enzyme. These that the T59R mutation has a on the thermal stability of the whereas the T59A mutation has almost no on the thermal of the and was used to the metal of the wild-type and mutant This on tightly bound metals, the enzyme is in buffer in for the The wild-type and T59A enzymes were found to have of zinc and and of magnesium of The indicated that there were levels of both magnesium and zinc in the T59R enzyme. These results that the T59R enzyme not zinc or magnesium or that these metals that the used in the of the enzyme The two active sites in the dimeric E. coli alkaline phosphatase are Å no active site residues are from the subunit, no obvious for obligatory mutants of the E. coli enzyme have been isolated that exhibit A form of the containing mutations in the two activity be upon the activity of the homodimeric used to the enzymes (12Schlesinger M.J. Levinthal C. J. Mol. Biol. 1963; 7: 1-12Google Scholar, M.J. A. Barrett Levinthal C. J. Mol. Biol. Scholar, A. S. J. Mol. Biol. 1963; 7: Scholar, M.J. Kantrowitz E.R. J. Mol. Biol. 2000; Scholar, M.J. A. Levinthal C. Biol. 1963; Scholar). This that the formation of the dimer interface in structural that activity. most APs have quaternary structures that are dimeric or of there are a APs to be such as from S. D. J. J. Scholar), Biochem. Biochem. Mol. Biol. Scholar), B. 2000; 27: Scholar), 1998; Scholar), and of the two APs from C. Jensen K. J. Scholar). In some at the evidence for the monomeric of these APs is In has been that the P. AP not exist as an active monomer stable monomers can be isolated under conditions. (15Martin D.C. Pastra-Landis S.C. Kantrowitz E.R. Protein Sci. 1999; 8: 1152-1159Google Scholar) have to the dimer interface of AP using mutations that hydrogen-bonding interactions subunits. some of the AP mutants were found in a of the mutations the interface to the isolation of a monomer form of the enzyme. In this an amino acid with a charged side chain was introduced into a of the interface that the dimeric form of the enzyme. The of a monomeric form of AP will to understand better the role of the dimer interface for the of E. coli AP. of a Alkaline studies of the interface of AP suggested a of that were for the of a and charged side chain that the formation of the dimeric structure. such was Thr-59, which is near the 2-fold axis of The of with two residues in the interface to each other in addition to to the interface. Analysis of the T59R AP indicated that the mutation the quaternary structure of the enzyme. The T59R enzyme was found by sucrose-density gradient to as for a monomer. The molecular of the T59R enzyme was determined to be that of the wild-type enzyme by a of sucrose-density gradient and size-exclusion no were between the for the monomer and dimer in the sucrose gradient of the T59R subunit association must be in the T59R enzyme. the T59R the T59A AP as a dimer, that the absence of replaced a was not to In the charged side chain of at is to the interface and dimer the T59R mutation monomer formation by altering the secondary structure of the circular dichroism was used to the T59A, T59R, and wild-type The monomer form of the T59R enzyme the secondary structural as the wild-type enzyme. The of these three APs were almost that the mutations at do not alter the secondary structure of the enzyme. Therefore, the of the of the T59R enzyme to dimerize is not the of in the secondary structure of the enzyme to the of the Alkaline The monomer form of alkaline phosphatase in this has catalytic activity and structural stability compared with the wild-type enzyme. The thermal melting for the as determined by CD, an change in thermal that E. coli is not a E. coli AP is an stable protein for its with a Tm of 97 °C. The more 50 °C reduction in Tm of the monomeric T59R AP 43 that the presence of the dimer interface structural to the enzyme. of the of the interface in E. coli AP is to thermal stability for the enzyme. It is to that of the alkaline to exist as a monomer was isolated from B. 2000; 27: Scholar), a The formation of quaternary structures well a mechanism for thermal stability for AP in and enzymes in The monomeric T59R AP has catalytic In the of the T59R AP is that by mutant of AP in which the residue has been replaced by or B. M.J. C. Kantrowitz E.R. J. Mol. Biol. 1998; Scholar). at required for the wild-type no significant in activity was These results that the activity of the T59R AP is not the of binding of the metals to the active site of the enzyme. the T59A enzyme has almost the as the wild-type the reduction in activity induced by the T59R mutation must be a direct of the quaternary structures of the T59A and T59R The which that the secondary structures of the T59A, T59R, and wild-type APs were this have been by these an subunit interface is required for catalytic of E. coli AP. stability of the subunit is upon association and formation of the dimer interface. found in of the structural of the interface has an role in dimer In the residues of the amino of AP have more This structural to that the of the protein is important for the dimeric structure. For example, of the 10 or residues from the amino the dimeric form of the E. coli enzyme and the activity by 20% (8Schlesinger M.J. Barrett K. J. Biol. Chem. 1965; 240: 4284-4292Google Scholar, R. J. Biol. Chem. Scholar). a monomeric and dimeric form of AP in C. Jensen K. J. Scholar, C. Wyckoff H.W. J. Biol. Chem. Scholar). The amino acid found between the B. AP and AP and the E. coli enzyme are and The and active site residues of the B. enzymes are well compared with E. coli AP. However, in the interface residues of the B. APs are less compared with E. coli AP. in the between the two B. and E. coli APs is residue coli C. Wyckoff H.W. J. Biol. Chem. Scholar). In the E. coli residue is a and in the dimeric B. AP, the corresponding residue is a whereas in the monomeric B. AP, the residue is The monomeric T59R E. coli can be compared with that of the monomeric B. enzyme the residue at is These together suggest that dimerization of the monomeric form of B. AP and of E. coli T59R AP not occur the side chain sterically This has of the functional and structural role of the AP such as the for which are for the formation of a catalytically functional active site The monomeric T59R AP bound amounts of magnesium or which are required in the catalytic mechanism of wild-type AP (7Stec B. Holtz K.M. Kantrowitz E.R. J. Mol. Biol. 2000; 299: 1303-1311Google Scholar). interactions are in the T59R the active site and this is in the activity. However, the of the interface interactions and formation of an active site do not the secondary the These suggest that the interactions of AP not the protein of AP the residues in the active sites that are required for and phosphatase activity. for plasmid