Báo cáo khoa học: Effects of replacing active site residues in a cold-active alkaline phosphatase with those found in its mesophilic counterpart from Escherichia coli

Effects of replacing active site residues in a cold-active alkaline phosphatase with those found in its mesophilic counterpart from Escherichia coli ´ sgeirsson Katrı´n Gudjo´nsdo´ttir and Bjarni A Department of Biochemistry, Science Institute, University of Iceland, Reykjavik, Iceland Keywords cold adaptation; metalloenzyme; mutagenesis; protein stability; psychrophilic Correspondence B A´sgeirsson, Science Institute, University of Iceland, Dunhaga 3, 107 Reykjavik, Iceland Fax: +354 552 89 11 Tel: +354 525 48 00 E-mail: bjarni@raunvis.hi.is (Received 26 September 2007, revised November 2007, accepted November 2007) doi:10.1111/j.1742-4658.2007.06182.x Alkaline phosphatase (AP) from a North Atlantic marine Vibrio bacterium was previously characterized as being kinetically cold-adapted It is still unknown whether its characteristics originate locally in the active site or are linked to more general structural factors There are three metal-binding sites in the active site of APs, and all three metal ions participate in catalysis The amino acid residues that bind the two zinc ions most commonly present are conserved in all known APs In contrast, two of the residues that bind the third metal ion (numbered 153 and 328 in Escherichia coli AP) are different in various APs This may explain their different catalytic efficiencies, as the Mg2+ most often present there is important for both structural stability and the reaction mechanism We have mutated these key residues to the corresponding residues in E coli AP to obtain the double mutant Asp116 ⁄ Lys274, and both single mutants All these mutants displayed reduced substrate affinity and lower overall reaction rates The Lys274 and Asp116 ⁄ Lys274 mutants also displayed an increase in global heat stability, which may be due to the formation of a stabilizing salt bridge Overall, the results show that a single amino acid substitution in the active site is sufficient to alter the structural stability of the cold-active Vibrio AP both locally and globally, and this influences kinetic properties Alkaline phosphatase (AP; EC 3.1.3.1) is a nonspecific catalyst for the hydrolysis or transesterification of phosphoryl esters, and usually functions best in an alkaline environment [1] AP from Escherichia coli has been most extensively studied [2], and its three-dimensional structure was the first to be determined [3,4] Various mutants of the E coli enzyme have been made [5–14], and crystal structures for many of these are known To date, structures of APs from only three other species have been solved, namely human placental AP [15], shrimp AP [16], and AP from an Antarctic bacterium [17] Sequence comparisons of APs from a variety of species combined with structural information suggest that functionally important domains are mostly conserved [3] The residues that directly react with the substrate, Ser102 and Arg166, are conserved in all cases In addition, there are three metal-binding sites, M1, M2, and M3, containing two Zn2+ (occupying M1 and M2) and one Mg2+ (in M3), in the active site of the E coli enzyme [3] All three metal ions participate in the reaction mechanism [18] Amino acid residues that interact with the zinc ions in the M1 and M2 sites of APs are conserved in all known sequences However, variations occur at amino acids Asp153 and Lys328 near the Mg2+-binding site (M3) of the E coli AP (Fig 1A) The only change observed at position 153 in other variants is from Asp to His, whereas position 328 is commonly changed from Lys to either Abbreviations AP, alkaline phosphatase; DGu, free energy of unfolding; pNPP, p-nitrophenyl phosphate; Tm, melting temperature; T50%, temperature required for the enzymes to lose half of the initial activity in 30 FEBS Journal 275 (2008) 117–127 ª 2007 The Authors Journal compilation ª 2007 FEBS 117 K Gudjo´nsdo´ttir and B A´sgeirsson Mutations in a cold-active alkaline phosphatase A B Fig (A) Active site of E coli AP and (B) active site of Vibrio AP The residues near the Mg2+ ion in the third metal-binding site of these two APs are shown The only variations between the two species are seen in positions 328 and 153 (E coli numbering) Lys328 and Asp153 are Trp274 and His116 in the Vibrio AP In the E coli enzyme, a salt bridge connects Lys328 and Asp153 Lys328 has been shown to be important for phosphate binding, and the type of amino acid in this position has been shown to affect what metal occupies the M3 site and which metals the enzyme can use for catalysis Residues that ligate the two Zn2+ are conserved in all APs and are not shown The position of the substrate phosphate leaving group is shown, as well as the Arg166 ⁄ 129 residue near the substrate-binding site The figures were generated using the PYMOL viewer The E coli model was (A) is made using the Protein Data Bank file 1ALK The Vibrio model is a homology model made with SWISS-PDBVIEWER 118 Trp or His Asp153 in the E coli enzyme ligates the Mg2+ and the substrate’s phosphoryl group through water molecules Lys328 has also been shown to be important for binding of phosphate in the active site of the E coli enzyme through a water-mediated link [9,19] Furthermore, these two amino acids, Asp153 and Lys328, are connected by a salt bridge in the E coli enzyme [3,9] It has also been shown that the type of amino acid at position 328 has an effect on which metal occupies the M3 site [10,11] and which metals the enzyme can use for catalysis [14,20] Experiments have shown that the presence of Mg2+ in M3 in E coli AP is important for full catalytic activity and for stability of the enzyme [9,21,22] Information about the reaction mechanism of APs comes mostly from studies on the E coli enzyme, but it is believed to be generally the same for all APs The reaction starts with a nucleophilic attack of a Ser102 alkoxide on the phosphate group of the phosphomonoester substrate, forming the covalent phosphoenzyme intermediate The second step is the hydrolysis of this phosphoseryl intermediate to the noncovalent enzyme–phosphate complex [23] In the presence of a phosphate acceptor, such as Tris or diethanolamine, the enzyme also displays transphosphorylating activity, an organic nucleophile replacing water [24] The ratedetermining step of the E coli enzyme reaction is pHdependent At acidic pH, the hydrolysis of the covalent phosphoseryl intermediate is the slowest step, but at basic pH, the dissociation of the phosphate from the noncovalent enzyme–phosphate complex becomes ratedetermining [25,26] The type of amino acid at position 328 in the E coli AP has been shown to affect the affinity for inorganic phosphate [11,19] and thus the rate-determining step The E coli AP mutants K328H, K328A and K328C have been made and characterized [27,28] All of these mutants had an altered rate-determining step as compared to the wild-type enzyme That is, at pH 8.0, the hydrolysis of the covalent phosphoseryl intermediate was the slowest step The mutants also had lower affinity for inorganic phosphate Psychrophilic organisms have adapted to low-temperature environments by making enzymes with better catalytic efficiency at low temperatures (higher kcat or kcat ⁄ Km) The common features of such coldactive enzymes as compared to their mesophilic counterparts are believed to be the consequence of enhanced structural flexibility, often leading to decreased thermostability [29–32] It is not clear whether increased flexibility is brought about by local weakening of stabilizing bonds around the active site, or by more general structural factors, and FEBS Journal 275 (2008) 117–127 ª 2007 The Authors Journal compilation ª 2007 FEBS K Gudjo´nsdo´ttir and B A´sgeirsson ln (ν/ν 0) –0.5 –1 –1.5 35 °C –2 –0.5 ln (ν/ν 0) it may be attained differently within different enzyme families [32] An AP was previously isolated from a costal water Vibrio bacterial strain that was found to be a kinetically cold-adapted 55 kDa monomer [33] Most other known APs are dimers composed of identical subunits, but other apparent monomeric APs have been reported Examples include AP from Vibrio cholera [34], the heat-labile APs from a Shewanella sp [35], and the AP from an Antarctic bacterium [36] In comparison to the E coli AP, the Vibrio AP had higher catalytic efficiency as well as considerably less structural stability, being sensitive to room temperature The Asp153 ⁄ Lys328 pair in E coli AP, important for substrate binding and the identity of the metal in M3 [11], is His116 ⁄ Trp274 in Vibrio AP (Fig 1B) This may explain their different catalytic efficiencies We have mutated Trp274 in Vibrio AP to Lys and His116 to Asp We have also made the double mutant Asp116 ⁄ Lys274, which is an analog of the E coli enzyme with regard to amino acids in the active site Our aim was to determine whether these amino acids take part in the cold adaptation of the Vibrio AP, and we found they clearly affect both stability and activity in a reciprocal manner Mutations in a cold-active alkaline phosphatase –1 –1.5 Results 45 °C Heat stability of the active site conformation The rate of heat inactivation was determined at several temperatures for wild-type Vibrio AP and the three mutants The results for 35 C and 45 C are shown in Fig Rate constants were determined and used to obtain t1 ⁄ values (Table 1) Furthermore, the temperature required for the enzymes to lose half of the initial activity in 30 (T50%) was also calculated, and the melting temperature (Tm) values were obtained by CD, as described in the next section (Table 2) The wildtype Vibrio AP was very sensitive to heat inactivation, confirming our previous results [33] Thus, the t1 ⁄ at 25 C was only 94 (Table 1), and T50% was 40.1 C (Table 2) In contrast, all three mutants were stable at 25 C for at least 30 (Table 1) At 55 C, the double mutant H116D ⁄ W274K lost half of its initial activity at min, but was more stable than either the W274K mutant (t1 ⁄ of 1.2 min) or the H116D mutant (t1 ⁄ of 0.2 min) Interestingly, the H116D mutant was slightly less heat-stable than the wild-type in terms of the T50% values (38.5 C versus 40.1 C), whereas both the W274K and H116D ⁄ W274K mutants were more heat-tolerant than the wild-type enzyme (Table 2) –2 10 15 20 Time (min) 25 30 Fig Heat inactivation of wild-type Vibrio AP (black squares), the H116D mutant (open squares), the W274K mutant (black circles) and the H116D ⁄ W274K double mutant (open circles) at 35 C (upper panel) or 45 C (lower panel) Samples were incubated at the indicated temperatures, and residual activity was measured at intervals of a few minutes using the standard enzyme assay m ⁄ m0 = relative activity Effect of heat on secondary structure The effect of heat on the secondary structures of the wild-type and mutant Vibrio APs was measured by CD at several temperatures Figure shows CD spectra of the wild-type enzyme at temperatures from C to 90 C Changes in secondary structures during heating from 15 C to 90 C were further monitored at 222 nm for the wild-type and all the mutants (Fig 4) From these data, Tm values were determined (Table 2) The double mutant had the highest Tm value, at 58.6 C The wild-type had a Tm of 56.5 C, slightly higher than that of the H116D mutant, which had a Tm of 55.0 C The value for the W274K mutant was 57.2 C FEBS Journal 275 (2008) 117–127 ª 2007 The Authors Journal compilation ª 2007 FEBS 119 K Gudjo´nsdo´ttir and B A´sgeirsson Mutations in a cold-active alkaline phosphatase T (C) Wild-type, t1 ⁄ (min) W274K, t1 ⁄ (min) H116D, t1 ⁄ (min) H116D ⁄ W274K, t1 ⁄ (min) 20 25 30 35 40 45 50 55 60 ND 94 ± 79 ± 31 ± 12 ± 2.3 ± 0.7 ± – – ND ND ND ND 231 26 1.2 – ND ND 2600 63 30 1.1 0.8 0.2 – ND ND ND 2200 217 96 10 5.0 0.5 1.1 0.2 ± ± ± ± 12 0.1 ± ± ± ± ± ± 140 0.3 0.2 0.1 Tm (C) H116D ⁄ W274K mutant W274K mutant Wild-type Vibrio AP H116D mutant 58.6 57.2 56.5 55.0 ± ± ± ± ± ± ± ± ± ± 0.8 0.6 0.4 195 19 0.3 0.1 20 30 40 50 60 T (°C) 70 80 90 Fig Effect of heat on secondary structure stability of the wildtype Vibrio AP and the H116D, W274K and H116D ⁄ W274K mutants The enzymes were heated from 15 C to 90 C at a rate CỈmin)1, and ellipticity at 222 nm was monitored and plotted at 0.1 C intervals Ellipticity at 222 nm has been normalized to fraction of unfolded protein The protein concentration was 0.06 mg ⁄ mL The sample buffer was 50 mM Mops and mM MgSO4 (pH 8.0) Urea denaturation of wild-type Vibrio AP and free energy of unfolding T50% (C) 0.2 0.1 0.2 0.3 47.0 44.7 40.1 38.5 ± ± ± ± 0.2 0.1 0.2 0.3 –5 CD (mdeg) Wt H116D W274K H116D/W274K 0.2 Table Heat stability of the Vibiro AP mutants Tm values were determined by measuring CD at 222 nm with continuous heating from 15 C to 90 C at CỈmin)1 T50% values were determined by following inactivation at various temperatures, and refer to the temperature at which the enzyme loses 50% of initial activity at 30 (n = 3) Enzyme Fraction unfolded Table Heat inactivation of the Vibrio AP variants at different temperatures Samples were incubated at the indicated temperatures, and residual activity was measured at intervals of a few minutes using a standard enzyme assay The t1 ⁄ values were calculated using the relationship t1 ⁄ = ln(2) ⁄ k, where k is the rate constant of inactivation at the appropiate temperature (n = 3) ND, no detectable activity loss over 30 –, activity loss too fast to measure °C 20 °C The effect of urea on wild-type Vibrio AP activity and tertiary structure was determined by p-nitrophenyl phosphate (pNPP) hydrolysis and fluorescence measurements (Fig 5) The results showed that loss of activity occurred at urea concentrations below m, before any change was detected in the global protein structure as monitored by tryptophan fluorescence The global structure was unfolded at urea concentrations in the range 1–3 m ([urea]1 ⁄ = 1.7 m) Free energy of unfolding (DGu) was calculated from these data using the linear extrapolation method [37] DGu for unfolding (measured by fluorescence) was 3.2 kcalỈmol)1, but it was 2.0 kcalỈmol)1 for the inactivation transition 40 °C –10 Zn2+ content and release from active site of wild-type Vibrio AP 50 °C 60 °C 80 °C –15 200 210 220 230 λ (nm) 240 250 Fig Heat-induced unfolding of wild-type Vibrio AP secondary structures monitored by CD measurements The enzyme was heated from C to 90 C at a rate of CỈmin)1, and CD spectra from 200 to 250 nm were taken every 10 C Selected samples are shown The sample buffer was 50 mM Mops and mM MgSO4 (pH 8.0) 120 The metal content of wild-type Vibrio AP was analyzed by inductively coupled plasma MS and atomic absorption Only Zn was consistently found in the protein peak in significant amounts by MS after desalting by column chromatography Very insignificant amounts of tin, cadmium, nickel, vanadium and cobalt were inconsistently seen coeluting with the protein peak in the various batches of samples that were analyzed The molar ratio of metals in a monomeric Vibrio AP determined FEBS Journal 275 (2008) 117–127 ª 2007 The Authors Journal compilation ª 2007 FEBS K Gudjo´nsdo´ttir and B A´sgeirsson Mutations in a cold-active alkaline phosphatase 355 80 350 60 345 40 λ max (nm) were incubated at 18 C either with no Mg2+ in the solution or with 10 mm Mg2+ added (Fig 6) With 10 mm Mg2+, the enzyme activity was completely stable over 160 However, with no Mg2+ in solution, the activity was lost quite quickly and had dropped to 20% of the initial activity after 160 At higher temperatures, the inactivation was very fast Kinetic properties 20 340 [Urea] (M) Fig Effect of urea on activity (open squares) and tryptophan fluorescence (black squares) of wild-type Vibrio AP The protein concentration was 0.01 mg ⁄ mL Samples were incubated for h at 15 C in 25 mM Mops and mM MgSO4 (pH 8.0) and different concentrations of urea before measurement of each spectrum Fluorescence is given as maximum emission wavelength (kmax) The excitation wavelength was 290 nm, and fluorescence was scanned from 310 to 400 nm at 15 C Residual activity was measured using the standard enzyme assay by atomic absorption spectrometry was determined as 1.9 ± 0.6 (n = 12) for zinc and 4.1 ± 1.7 (n = 6) for magnesium No cobalt or copper was detected Thus, wild-type Vibrio AP most likely has two Zn2+ and one Mg2+ in the active site in addition to three Mg2+ that bind elsewhere Evidence for additional Mg2+-binding sites in APs, possibly with a functional role, is emerging from known crystal structures [17] Zn2+ release from the active site of wild-type Vibrio AP was monitored while the enzyme was inactivated by either heat or urea treatment The chelator 4-(2-pyridylazo)-resorcinol was used to detect free Zn2+ At 37 C, the enzyme lost activity quite quickly, but no Zn2+ release was detectable over a period of h At 60 C, the enzyme released two equivalents of Zn2+ within 30 At that temperature, inactivation of the enzyme was spontaneous Similar results were obtained with urea denaturation experiments No Zn2+ release was observed at urea concentrations below m, whereas at that concentration all activity had been lost (data not shown) The results suggest that Zn2+ release from the active site coincides with total protein structure unfolding but that the activity is lost before that event Effect of Mg Vibrio AP 2+ on active site stability of wild-type The effect of Mg2+ concentration on wild-type Vibrio AP enzyme activity was investigated Enzyme samples The catalytic efficiencies of wild-type Vibrio AP and the three mutants were determined both under hydrolysing and under transphosphorylating conditions (Tables and 4, respectively) Kinetic constants for the E coli AP were also measured for comparison Under conditions in which the phosphoseryl intermediate is hydrolyzed (Table 3), the wild-type Vibrio AP had the highest catalytic efficiency (kcat ⁄ Km), 10-fold higher than that of the E coli enzyme All enzyme activities were measured at their optimal pH under hydrolyzing conditions (pH 8.0 for the E coli enzyme and pH 9.8 for the Vibrio enzymes) All the mutants had much reduced catalytic efficiencies as compared to the wild-type Vibrio enzyme The kcat for the W274K mutant was seven times lower than that for the wildtype (changing from 1580 s)1 to 220 s)1) The Km of that mutant was also increased from 0.08 mm for the wild-type enzyme to 0.19 mm Overall, the catalytic efficiency of the W274K mutant was 16.5 times lower 100 80 Relative activity (%) Relative activity (%) 100 60 40 20 0 20 40 60 80 100 Time (min) 120 140 160 Fig Effect of Mg2+ on wild-type Vibrio AP active site stability Wild-type Vibrio AP in 10 mM Mg2+ solution (black squares) and wild-type Vibrio AP in a solution without added Mg2+ (open squares) The background solution was 20 mM Tris and 15% ethylene glycol (pH 8.0) Samples were incubated at 18 C Residual activity was measured using the standard enzyme assay FEBS Journal 275 (2008) 117–127 ª 2007 The Authors Journal compilation ª 2007 FEBS 121 K Gudjo´nsdo´ttir and B A´sgeirsson Mutations in a cold-active alkaline phosphatase Table Kinetic constants for the native Vibrio AP and its mutants as compared to E coli AP under hydrolyzing conditions Conditions were 50 mM Mops, 50 mM CAPS, and mM MgSO4, using pNPP as substrate at the optimum pH for each enzyme (pH 8.0 for the E coli AP and pH 9.8 for the Vibrio variants) Each number is the average of at least three independent experiments E coli AP Wild-type Vibrio AP Vibrio W274K mutant Vibrio H116D mutant Vibrio H116D ⁄ W274K mutant kcat (s)1) Km (mM) 38 1580 220 830 930 0.021 0.08 0.19 1.25 1.44 ± ± ± ± ± 132 16 50 86 ± ± ± ± ± 0.002 0.01 0.02 0.03 0.03 kcat ⁄ Km (s)1ỈmM)1) Ki (for Pi) (mM) 1900 19 750 1157 664 645 0.045 0.39 0.77 5.25 3.45 ± ± ± ± ± 0.011 0.05 0.11 0.71 0.28 Table Kinetic constants for the native Vibrio AP and its mutants as compared to E coli AP under transphosphorylating conditions Conditions were 1.0 M Tris, mM MgSO4 and pH 8.0 with pNPP used as substrate Each number is the average of at least three independent experiments E coli AP Wild-type Vibrio AP Vibrio W274K mutant Vibrio H116D mutant Vibrio H116D ⁄ W274K mutant kcat (s)1) Km (mM) kcat ⁄ Km (s)1ỈmM)1) Ki (for Pi) (mM) 89 2050 1940 1380 1830 0.06 0.13 0.63 1.43 2.20 1483 15 769 3079 965 832 0.07 0.10 1.63 0.77 1.43 ± ± ± ± ± 10 131 162 98 72 than that of the wild-type The H116D and the H116D ⁄ W274K mutants also had much reduced turnover rates, and for both of these enzymes, substrate binding was greatly impaired (15–18 times greater Km) The wild-type enzyme was a 30-fold better catalyst (kcat ⁄ Km) than these two mutants under hydrolyzing conditions Although the E coli enzyme had a much lower turnover rate than the three Vibrio mutants, it was overall a slightly better catalyst, due to tighter substrate binding The E coli enzyme also had the lowest Ki for inorganic phosphate as compared to the wild-type Vibrio AP, indicative of higher binding affinity for the competitive inhibitor Binding affinities of the H116D and H116D ⁄ W274K mutants were especially low for the same reason Under transphosphorylating conditions, the enzymes were compared at pH 8.0 (the pH optimum for the E coli enzyme) The Vibrio wild-type AP was still the best catalyst of the five enzymes, although it was not at its optimum pH, with 10-fold better catalytic efficiency than the E coli AP Under these conditions, the Vibrio mutants showed insignificantly lower turnover rates as compared to the wild-type, except for the H116D variant However, reduced substrate binding of all three mutants made them much less efficient catalysts For the wild-type Vibrio AP, the turnover rate did not increase much when going from hydrolyzing to transphosphorylating conditions However, the three Vibrio AP mutants, especially the W274K variant, seemed to be much more dependent on the organic phosphate 122 ± ± ± ± ± 0.02 0.03 0.02 0.12 0.19 ± ± ± ± ± 0.01 0.02 0.09 0.04 0.09 acceptor in attaining high turnover rates, an indication that breakdown of the phosphoseryl intermediate is the rate-limiting step in the cold-active AP variants Discussion Stability of wild-type Vibrio AP and the mutants The active site area of wild-type Vibrio AP was very heat-sensitive At a temperature as low as 25 C, the t1 ⁄ of the enzyme’s activity was only 94 At that temperature, no activity loss was detected for the three mutants produced here H116D began to lose activity rapidly at 35 C, but the W274K and the H116D ⁄ W274K mutants were stable up to 40 C However, the heat stability of APs from E coli and other mesophilic bacteria is even greater [1] The E coli enzyme is especially heat-tolerant, with a t1 ⁄ of at 90 C [1], and AP from the mesophilic Bacillus subtilis has a t1 ⁄ of 28 at 65 C Only one AP has been characterized that is more heat-labile than the Vibrio AP, the AP from the Antarctic bacterium HK47 [36], with a t1 ⁄ of only at 40 C as compared to for the Vibrio AP at the same temperature Other heat-labile and coldactive APs displayed t1 ⁄ values of 15 at 45 C [38] and 3–30 at 55–65 C [35,39,40] From these findings, it may be concluded that the Vibrio AP is a good example of a cold-adapted AP The effect of heat on activity is often used as an indication of the total heat stability of enzyme structures FEBS Journal 275 (2008) 117–127 ª 2007 The Authors Journal compilation ª 2007 FEBS K Gudjo´nsdo´ttir and B A´sgeirsson For the wild-type Vibrio AP, however, loss of activity and loss of secondary structures did not coincide Activity was lost at much lower temperatures than the changes in secondary structures observed by CD Urea denaturation of the wild-type enzyme gave similar results (Fig 5), indicating local vulnerability in the active site area Structural stability (Tm obtained by CD) was increased for the W274K and the H116D ⁄ W274K mutants by 0.7 C and 2.1 C, respectively, as compared to the wild-type enzyme Interestingly, however, the H116D variant showed decreased structural stability, and Tm was lowered by 1.5 C A correlation was observed whereby for the two more stable variants (W274K and H116D ⁄ W274K), the more robust global structure brought increased stability to the active site area, whereas for the H116D variant, both factors were reversed as compared with the wild-type structure We may conclude that the active site of the cold-active Vibrio AP has evolved to be especially flexible and consequently more heat-intolerant than the whole structure of the enzyme A possible explanation for the increased heat stability of the W274K variant might stem from the fact that lysine is able to form ionic bonds with an adjacent negatively charged amino acid and forms more stabilizing bonds in the active site than tryptophan does, perhaps for steric reasons Tryptophan has some preference for binding sites where large conformational changes occur, causing hydrophobic–hydrophobic, aromatic– aromatic and hydrophobic–polar residue pair interactions [41] Inspection of the homology model revealed several possibilities for bonding with W274 These include Q18, H286, H316, and E317 Furthermore, bond networks mediated by water molecules could extend the crosslinking possibilities Further speculation regarding individual interactions is not warranted until a precise structure has been solved Our experimental results showed that the E coli AP mimic H116D ⁄ W274K was indeed the most stable of the mutants made here, with an increased Tm of 2.1 C as compared to the wild-type Vibrio AP In the E coli AP, a salt bridge exists between Asp153 and Lys328 [3] (corresponding to amino acids 116 and 274 in the Vibrio AP) It may be that such a salt bridge has also formed in the H116D ⁄ W274K mutant and gives the enzyme increased stability, as was observed However, it was a long way from equalling the reported Tm of E coli AP of up to 97.0 C [1,42] The 2.1 C increase in Tm for the H116D ⁄ W274K mutant is clearly a result of the favorable combination of the two single mutations, as the H116D mutant had a reduction in Tm of 1.5 C, and the W274K mutant showed an increase in Tm of 0.7 C The absence of an ionic bond involving Mutations in a cold-active alkaline phosphatase amino acids 116 and 274 in the wild-type Vibrio AP may thus represent a part of the enzyme’s cold adaptation Catalytic efficiency The catalytic efficiency of wild-type Vibrio AP is 10 times greater than that of the E coli enzyme, both under hydrolyzing and under transphosphorylating conditions Km was higher for all the mutants, and the turnover number was reduced Furthermore, all three mutants studied here had higher Ki values for the competitive inhibitor inorganic phosphate, indicating reduced binding affinity If the rate-determining step of the Vibrio AP reaction is the release of the phosphorous product from the noncovalent enzyme–phosphate complex, as found for the E coli AP [25,26], lower affinity for inorganic phosphate should lead to an increase in the catalytic rate (kcat) in the mutants Such an increase was not seen, which points to a different rate-determining step for the Vibrio AP as compared to the E coli enzyme Deacylation would be the most likely candidate, perhaps involving a conformational change [23,43] The conformational change needed for the deacylation step may be more easily achieved in cold-active enzymes such as the Vibrio AP, as they are believed to have more flexible structures than their mesophilic counterparts The Km of the W274K mutant was increased threefold as compared to the wild-type enzyme under both assay conditions examined (Tables and 4) In the E coli enzyme, the equivalent Lys328 makes a watermediated connection to inorganic phosphate [3] that the Lys in the Vibrio AP W274K mutant apparently does not make, because it showed reduced affinity both for the substrate and for inorganic phosphate as compared to the wild-type enzyme The kinetic constants of a W274A mutant (data not shown) were nearly identical to those of the W274K mutant, supporting this conclusion This is in contrast to the position of Lys328 in the D153H mutant of E coli AP [9], which is an analog of our W274K mutant with respect to residues 153 and 328 Affinity for the substrate and inorganic phosphate was increased in the D153H mutant as compared to the wild-type E coli AP, possibly due to loss of the salt bridge between the Asp normally in position 153 and Lys328 When no ionic connection was present, it was suggested that the Lys would change position and connect directly to the phosphorous group, leading to tighter binding of both substrate and inhibitor Although Lys is probably positively charged in the Vibrio AP active site environment, and could therefore make a connection to FEBS Journal 275 (2008) 117–127 ª 2007 The Authors Journal compilation ª 2007 FEBS 123 K Gudjo´nsdo´ttir and B A´sgeirsson Mutations in a cold-active alkaline phosphatase inorganic phosphate, as may be the case in the D153H E coli mutant, it does not appear to so It is possible that it points away from the substrate and forms an ionic connection to some negatively charged amino acid elsewhere Results similar to ours were obtained for the H317K mutant of human placental AP [44], which is also an analog of our W274K mutant In that case, substrate affinity was reduced in an H317K mutant, suggesting a worse connection of a Lys in this position to the phosphorous moiety of the substrate, leading to a conclusion similar to ours: namely, that the position of Trp274 in Vibrio AP does not allow it to make stabilizing interactions with bound phosphate groups of equivalent strength to that which produces the low Km of the E coli AP even when it is changed to a Lys As compared to wild-type Vibrio AP, the H116D single mutant had highly reduced affinity for the substrate and the inhibitor inorganic phosphate, as judged by Km and Ki values (Tables and 4) This may be a result of the negative charge of the Asp repelling the negative charges of both substrate and inhibitor The K328W mutant of E coli AP is an analog of our Vibrio AP H116D mutant with respect to those two sequence positions That enzyme also showed reduced substrate affinity as compared to wild-type E coli AP [14], as did AP from Saccharomyces cerevisiae, which also has Asp in the corresponding position [45] All these results show that the presence of an Asp residue in this position (residue 153 according to E coli numbering) without a Lys residue in the position corresponding to residue 328 in E coli causes less affinity for both substrate and the inhibitor, inorganic phosphate Finally, the H116D ⁄ W274K Vibrio AP double mutant is the active site analog of the wild-type E coli AP, where there is an ionic connection between Asp153 and Lys328 [3] The H116D ⁄ W274K Vibrio AP mutant needed very high concentrations of substrate or inhibitor as compared with the wild-type or the single mutants A possible explanation for the observed characteristics could be a combination of two things: repellent forces between the negative charge on Asp116 and negative charges on substrate and inhibitor, and the reduced connection of Lys274 to the substrate and inhibitor as compared to Trp274, due to lack of the expected salt bridge and poorer positioning Heat stability measurements point to additional ionic connections being formed involving Asp116 and Lys274 (Figs and 4, and Tables and 2) Given that it is unlikely, due to distance, that the Lys–Asp salt bridge is formed in the H116D ⁄ W274K mutant, the reduced substrate ⁄ inhibitor affinities of this enzyme may be 124 explained by altered electrostatics and possibly increased flexibility of the active site A structural constriction resulting from new crosslinking that impedes the chemical step(s) may be postulated to explain lower turnover rates of this mutant as compared to the wildtype Vibrio AP, making its catalytic and stability properties change in the direction of the E coli counterpart without fully reaching the properties of the latter Experimental procedures Materials Salts and general chemicals were obtained either from Sigma (St Louis, MO, USA) or Merck (Darmstadt, Germany) l-Histidyldiazobenzylphosphonic acid on agarose, p-nitrophenyl phosphate (pNPP), ampicillin, SDS, E coli AP (P-5931), lysozyme and deoxyribonuclease were also obtained from Sigma PhastSystem 8–25% polyacrylamide gels, SDS buffer strips, Coomassie Brilliant Blue for PhastSystem and MonoQ ion exchange columns were purchased from Amersham Pharmacia-Biotech (Uppsala, Sweden) Tryptone and yeast extract were purchased from Difco Laboratories (Detroit, MI, USA) Restriction endonucleases Apa1, Dpn1, HindIII and PstI, as well as DNA T4 ligase, DNA size standards, SDS protein standards, and LB agar, were obtained from Fermentas (Burlington, Canada) Agarose was purchased from FMC Bioproducts (Rockland, ME, USA) Plasmid pBluescript II SK (+) and the QuikChange mutagenesis kit were obtained from Stratagene (La Jolla, CA, USA) Oligonucleotide primers were obtained from T-A-G Copenhagen (Copenhagen, Denmark) Chemicals for sequencing were obtained from Applied Biosystems (Foster City, CA, USA) Gene cloning The Vibrio AP gene was cloned from a pUC18 plasmid that had been previously inserted [46] into the vector pBluescript KS II (+) Construction of mutants Mutants of Vibrio AP were constructed with a QuikChange mutagenesis kit, according to the manual from the manufacturer (Stratagene) PCR was performed in a GeneAmp-PCR system 2700 from Applied Biosystems Pfu polymerase (Stratagene or Fermentas) was used for the reactions Chemically competent E coli TOP10 cells were transformed with the reaction product Colonies were cultured and plasmid DNA was isolated from the cells using the Qiaprep Spin Miniprep Kit from Qiagen Those plasmids were sequenced, and one of the plasmids containing the desired mutation was used for the transformation of FEBS Journal 275 (2008) 117–127 ª 2007 The Authors Journal compilation ª 2007 FEBS K Gudjo´nsdo´ttir and B A´sgeirsson competent E coli cells of strain LMG194 (Invitrogen) that lack native AP [F–, DlacX74, galE, thi, rpsL, DphoA, (PvuII), Dara714, leu::Tn10] Mutations in a cold-active alkaline phosphatase double mutant [47] The protein concentration for E coli AP enzyme solutions was estimated by measuring absorbance at 278 nm and using the absorbance coefficient 0.71 cm2Ỉmg)1 [48] Expression Expression was performed in L portions (divided into nine L flasks) LMG194 cells containing the plasmid encoding the Vibrio AP enzyme or mutants were cultured in LB medium supplemented with 100 lgỈmL)1 ampicillin Cells were grown at 18 C with shaking (220 cyclesỈmin)1) Cell density was monitored by measuring A600 Cells were further handled for protein extraction 10–12 h after they had reached stationary phase Extraction of protein from cells Cultured cells were centrifuged for 10 at 6000 g using a Sorvall RC5C centrifuge The precipitated cells were then redissolved in : 10 of the original volume in 0.01% Triton X-100, 0.5 mgỈmL)1 lysozyme, 20 mm Tris, 10 mm MgCl2 (pH 8.0) The solution was left to stand for 2–3 h, and then frozen at – 20 C After freezing, the solution was thawed, DNase was added to a final concentration of 0.05 lgỈmL)1, and the resulting solution was left to stand for 30 at room temperature The solution was then centrifuged at 10 000 g for 15 at C Active AP was now in a clear solution Protein purification The solution containing active Vibrio AP was purified on an l-histidyldiazobenzylphosphonic acid agarose column at C as previously described [33] The enzyme was further purified and concentrated on a MonoQ ion exchange column connected to an FPLC apparatus (Amersham Pharmacia-Biotech) maintained at 18–20 C The enzyme was eluted with a 0–0.7 m NaCl gradient Enzymatic assay Enzyme activity was routinely measured with mm pNPP in 1.0 m diethanolamine buffer, containing 1.0 mm MgSO4, at pH 9.8 and 25 C Reactions were initiated by the addition of enzyme, and the release of p-nitrophenol (e = 18.5 m)1Ỉcm)1) was monitored at 405 nm Protein determination Protein concentration was estimated by measuring absorbance at 280 nm and using a calculated absorbance coefficient of 0.96 cm2Ỉmg)1 (e = 55 810 m)1Ỉcm)1) for the wild-type and H116D mutant, and 0.87 cm2Ỉmg)1 (e = 50 310 m)1Ỉcm)1) for the W274K mutant and the Kinetics Determination of kinetic constants was performed at seven different substrate concentrations in the range 0.04– 0.8 mm Kinetic constants were determined both under hydrolyzing and transphosphorylating conditions at 25 C The transphosphorylating conditions were mm Tris and mm MgSO4 (pH 8.0), and the hydrolyzing conditions were 50 mm Tris and 50 mm Caps (pH 9.8 or pH 8.0) Kinetic constants were calculated using the Lineweaver– Burk transformation of the Michaelis–Menten equation Kcat values were calculated from Vmax values using molecular masses of 58 kDa for the Vibrio phosphatases and 94 kDa for the E coli phosphatase [49] For determination of Ki for inorganic phosphate, Kmapparent for catalysis in the presence of 1.0 and 2.5 mm phosphate was measured, and Ki was calculated using Kmapparent = Km(1 + [I] ⁄ Ki) Temperature stability measurements For determination of active site thermal stability, enzyme samples were incubated at different temperatures in 20 mm Tris and 10 mm MgSO4 (pH 8.0), and activity was measured after different incubation times by the standard protocol A J-810 CD spectrometer from Jasco (Tokyo, Japan) was used to obtain CD spectra from 190 to 260 nm and determine Tm curves for the Vibrio APs Experiments were carried out in 50 mm Mops and mm MgSO4 (pH 8.0) For the Tm experiments, the CD signal at 222 nm was monitored as the temperature increased by CỈmin)1 The protein concentration was in the range 0.05–0.1 mgỈmL)1 Urea denaturing experiments Enzyme samples were incubated for h at 15 C in 25 mm Mops and mm MgSO4 (pH 8.0) solutions containing different concentrations of urea prior to measurements obtained by dilution from a m stock solution Residual enzyme activity was measured using the standard assay The intrinsic fluorescence of each sample was measured using a Fluoromax instrument and analyzed using Datamax software (Jobin Yvon) The excitation wavelength was 290 nm, and fluorescence was scanned from 310 to 400 nm at 15 C An average of three scans was taken For each sample, the maximum emission wavelength (kmax) was determined The protein concentration was 0.009– 0.011 mgỈmL)1 Background spectra were determined at each urea concentration and subtracted General data handling followed well-established procedures [37,50] FEBS Journal 275 (2008) 117–127 ª 2007 The Authors Journal compilation ª 2007 FEBS 125 K Gudjo´nsdo´ttir and B A´sgeirsson Mutations in a cold-active alkaline phosphatase Metal ion analysis Immediately before mass analysis, the samples were run at 0.6 mLỈmin)1 and room temperature through a HiTrap desalting column in an Agilent (Morges, Switzerland) 1200 Series apparatus, using 20 mm NH4H2PO4 (pH 8) as a mobile phase Monitored masses on an Agilent 7500ce inductively coupled plasma MS instrument were Mg, V, Mn, Co, Ni, Zn (66), Zn (68), Mo, Cd, and Sn For atomic absorption analysis, samples were dialyzed with three changes over a day period in Spectropor membrane tubing at C using a : 250 excess of Chelex-treated 20 mm Tris (pH 8.0) buffer The concentration of protein for analysis was 0.25–0.4 mgỈmL)1, and samples were acidified with HCl prior to injection A Varian (Crawley, England) Spectr220 FS atomic absorption spectrometer was employed to measure Zn, Mg, Co, and Cu 10 11 Acknowledgements The Icelandic Research Fund and the University of Iceland Research Fund supported this work financially We thank Dr Ernst Schmeisser for the mass analysis of metals, and Dr Sigridur Jonsdottir for assistance with atomic absorption analysis Professor Olafur S Andresson 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