1. Alkaline Phosphate Activity: The Removal and Reintroduction of Zinc and Magnesium Jesse Caballero, TJ Corley, Sherilyn Mumme, Joe Quiroz

2. Introduction

3. Introduction: Alkaline Phosphatase - Purple= Zn, Green= Mg, Orange= PO 4, Red= H 2 0 - Indigo= Serine, Cyan= Histidine, White= Aspartic acid - The purposed active site structure dependent on 2 Zinc and 1 Magnesium

4. Theory Alkaline Phosphatase (E.C. 3.1.3.1) from Escherichia coli exists within the periplasmic region – subject to elevated environmental constituents; therefore, retains structure under varying conditions. Proven to have increased resistance to: degredation, inactivation, denaturation, and intrinsically has a greater rate of activity. Alkaline Phosphatase, obtained from swine kidney, was inhibited by EDTA and cyanide due to the chelating removal of metal from active site H. Csopak et al. showed that in absence of EDTA, only two Zn 2+ were required for full enzyme activity. Furthermore, saturating AP in EDTA removed Zinc and Magnesium rendering inactive. – Also reintroduce Co 2+ producing activity within alkaline phosphatase but with a new degree of specificity. Alkaline phosphatase’s structural stability due to periplasmic location produced a protein that remained structurally unaltered when metal ions were removed; under specific conditions

5. Hypothesis Removal of active site metal ions would render alkaline phosphatase inactive; however, reactivation is obtained when metal ions are reintroduced into deactivated alkaline phosphatase. Since H. Csopak et al. achieved reactivation when experimenting with similar transition metals, it is possible to reactivate alkaline phosphatase with optimal metal ions to achieve the same rate of activity as unaltered AP Stec et al. purposed a three metal ion active site within AP – This claim was enhanced by Vallee et al. which indicated magnesium was ignored by previous experimentation, but aided in activity Wilson et al presented the capability of alkaline phosphatase to have four zinc-binding sites, which are not equivalent, but enhance Csopak et al.’s claim of only 2 zinc requirement for activity.

6. Secondary Experiment – Structural stabilizing role of Magnesium within Alkaline phosphatase increased rate of activity; however, is not directly involved in general base catalysis Herschlag et al. contradicted previous claims that third metal ion site, magnesium, provides general base catalysis. – Their results indicate the third metal ion stabilizes the transferred phosphoryl group within the transition state. – Removal of Mg 2+ effected both phosphate and sulfate monoester hydrolysis reactions; however, does not significantly effect phosphate diester hydrolysis – Magnesium does not mediate general base catalysis Observing the activity of Alkaline phosphatase after reactivation in varying metal concentrations will give insight to metal triad active site functionality

7. Overview of methods

8. Major Steps Changing of the original Alkaline Phosphatase buffer using dialysis Chelating of the Alkaline Phosphatase using EDTA and dialysis to remove the EDTA and metals Addition of Zinc and/or Magnesium for samples Activity assays of samples

9. Experimental Design Alkaline phosphatase was chelated over night by saturation with EDTA In activation of AP was confirmed with activity assay. Stoichiometric amounts of metals were added to 50 µL of inactivated enzyme for 24 hours at room temperature Enzyme assay was performed after over night incubation

10. Methods - Dialysis Dialysis exchanges the salts and other small molecules across a filtration membrane. Protein is eluted into a new buffer of workable sets of parameters for activity assays

11. Ethylenediaminetetraacetic acid (EDTA) EDTA is a excellent chelating molecule for metals such as Magnesium and Zinc The negative charges on the Oxygen stabilize the positively charged metal

12. Cofactors Metals provide Alkaline phosphatase with chemical properties that help carry out the hydrolysis of phosphate on a variety of substrates. In the wild type of Alkaline Phosphatase Zinc and Magnesium are found in stoichiometric ratios of 2 Zn and 1 Mg per molecule of enzyme

13. Activity Assay The enzyme of interest is a phosphatase so a substrate para- nitrophenol phosphate (PNPP) is particularly useful. The conjugated Pi-system of PNPP changes when the phosphate is hydrolyzed by AP at a wave length of 410nm The absorption of substrate is correlated to the activity of Enzyme

14. Extensive Materials and methods

15. Materials 108 mM Alkaline Phosphatase Tris/HCl Tris/Cl - Sorval Centrifuge Centrifuge dialysis tube and 40 kDa membrane EDTA MgCl 2 6H 2 O ZnCl 2 6H 2 O Cary 50 Spectrophotometer

16. Changing of the original Alkaline Phosphatase buffer: 75 µL of the stock Alkaline Phosphatase was added to 2425 µL of buffer in a centrifuge dialysis tube with a 40 kDa membrane. 2.5 mL of water was used on the opposite side of the centrifuge. The solution was run in a centrifuge for 30 minutes at 8,000 rpm. The supernatant was kept and the remaining solution was discarded. The supernatant was less than 10 µL. 1 mL of Tris buffer was added to the centrifuge dialysis tube with the supernatant and centrifuged for 10 minutes at 3,000 rpm. Again, the supernatant was kept and the remaining solution was measured and then discarded. 1 mL of Tris buffer was added to the 428 µL of the supernatant and the solution was centrifuged for 10 minutes at 3,000 rpm. 260 µL of the supernatant (which included the Tris buffer and the Alkaline Phosphatase) was collected after the dialysis. To get the supernatant off of the membrane it was centrifuged for 5 minutes at 3,000 rpm with the membrane upside down. 1 mL of Tris buffer at pH 7.4 was added to the supernatant in order to dilute and store the solution in 4°C. An activity assay was done on the resulting protein solution.

17. Chelating of the Alkaline Phosphatase and removal of EDTA: 1 mL of 0.1 M EDTA was added to 0.5 mL of the Tris/AP solutions. This mixture was allowed to come to equilibrium over 20 hours at room temperature. A dialysis was again carried out in order to remove the EDTA and the chelated metals. The solution was centrifuged for 30 minutes at 8,000 rpm. 1.41 mL of solution was removed. 1.5 mL of Tris buffer was added to the 90 µL of the denatured enzyme and the original Tris buffer. The centrifugation was carried out for 10 minutes at 3,000 rpm. 0.79 mL of the AP solution was recovered after the dialysis. 1 mL of Tris buffer was added and the solution was centrifuged for a final time for 10 minutes at 3,000 rpm. To get the supernatant off of the membrane it was centrifuged for 5 minutes at 3,000 rpm with the membrane upside down. 0.91 mL of the denatured Alkaline Phosphatase in Tris buffer was collected and used to run the samples. An activity assay was done on the solution containing the chelated protein.

18. Addition of Zinc and/or Magnesium: The Zinc and Magnesium were added according to stoichiometric ratios. 0.203 g of MgCl 2 was added to 10 mL of Tris buffer and 0.136 g of ZnCl 2 were added to 10 mL of Tris buffer. Then for each of the enzyme solutions, 1.8 µL of the metal solutions were added to the denatured enzyme/Tris buffer solution until a volume of 50 µL is reached. These samples were incubated at room temperature for 20 hours.

19. Samples The different mixtures were – 1 Zn 2+ : 0 Mg 2+ : 1 AP – 2 Zn 2+ : 0 Mg 2+ : 1 AP – 0 Zn 2+ : 1 Mg 2+ : 1 AP – 1 Zn 2+ : 1 Mg 2+ : 1 AP – 2 Zn 2+ : 1 Mg 2+ : 1 AP – 1 Zn 2+ : 2 Mg 2+ : 1 AP All ratios are according to stoichiometry

20. All of the activity assays were conducted in the following manner: 0.5 mL of 1 µM PNPP was added to 0.45 mL of the Tris buffer at pH 7.4. This solution was used as the blank. 50 µL of the enzyme solution was added and the enzyme kinetics at 410 nm was taken and analyzed with a molar absorptivity of 17500 M -1 cm -1. The change in absorption was collected for each of the different samples. Each of the samples differed in the concentration of the metals (Zn2 + or Mg 2+ ). 15 different mixtures were measured.

21. Results

22. Enzyme activity Activity 1 (µM/min) Activity 1 (µM/min) Activity 2 (µM/min) Activity 3 (µM/min) 1 Zn18.97784.95*55.67 2 Zn36.6768.78*60.8 1 Mg0.5721.00n/a 1 Zn 1 Mg23.8129.62n/a 2 Zn 1 Mg64.8571.71n/a 1 Zn 2 Mg44.2313.8460.43

23. Discussion

24. Dialysis A Dialysis was run initially to remove the contaminating metals, Mg 2+ and Zn 2+ To inactive AP, the chelation of Mg 2+ and Zn 2+ was necessary. This was accomplished by saturating the enzyme with EDTA. A second dialysis was required to remove EDTA. This yields a solution containing only the apoenzyme.

25. Activity Assay PNP - was the substrate used because AP is a phosphatase. PNP - provides absorption at 410 nm due to its conjugated Pi system Initially Velocity, V 0, was calculated by taking the change of the concentration of the AP at 410 nm and dividing by the change in time. The time interval was the first 10 seconds. The concentration of PNPP which was added completely saturated the enzyme, thus V 0 is the equivalent to V max. This provides consistency for measuring activity

26. Activity Assay The activity of AP after saturation of EDTA indicated successful chelation The activity of the chelated enzyme was 0.59 µM/min. This indicates that there was some enzyme left in the solution that was completed chelated; however, compared to the original activity (73 µM/min) this was significantly decreased.

27. Brownian motion of metal ion solutions Activation observed in varying solutions showed a potential ratio Zn 1 was less than Zn 2 because due to higher concentrations a greater amount of metal was able to be reintroduced into the AP Enzyme activity Activity 1 (µM/min) Activity 1 (µM/min) Activity 2 (µM/min) Activity 3 (µM/min) 1 Zn18.97784.9555.67 2 Zn36.6768.7860.8 1 Mg0.5721.00n/a 1 Zn 1 Mg23.8129.62n/a 2 Zn 1 Mg64.8571.71n/a 1 Zn 2 Mg44.2313.8460.43

28. Brownian motion of metal ion solutions Mg showed slight activation; however, since this is known to not be compatible with enzyme activity, the activity is compared to post- deactivation AP – The rate of activity of “deactivated” AP was comparative to Mg solution; therefore, EDTA was not successful at fully deactivating the enzyme and Mg was not able to increase activity alone Enzyme activity Activity 1 (µM/min) Activity 1 (µM/min) Activity 2 (µM/min) Activity 3 (µM/min) 1 Zn18.97784.9555.67 2 Zn36.6768.7860.8 1 Mg0.5721.00n/a 1 Zn 1 Mg23.8129.62n/a 2 Zn 1 Mg64.8571.71n/a 1 Zn 2 Mg44.2313.8460.43

29. Brownian motion of metal ion solutions Ratio between Zn 1 Mg 1 and Zn 2 Mg 1 in comparison to Zn 1 and Zn 2 – Increased enzyme activity; however, magnesium is known to not produce activity alone. Therefore, magnesium's enzyme functionality is aiding, not inducing activity Enzyme activity Activity 1 (µM/min) Activity 1 (µM/min) Activity 2 (µM/min) Activity 3 (µM/min) 1 Zn18.97784.9555.67 2 Zn36.6768.7860.8 1 Mg0.5721.00n/a 1 Zn 1 Mg23.8129.62n/a 2 Zn 1 Mg64.8571.71n/a 1 Zn 2 Mg44.2313.8460.43

30. Mechanism: The Three Metal Triad In the free enzyme, three water molecules fill the active site and the Ser102 hydroxyl group participates in a hydrogen bond with the Mg - coordinated hydroxide ion. HO-(Ser102) (Mg2+)-OH

31. 31 Mechanism: The Three Metal Triad Upon binding of the phosphomonoester, which forms a Michaelis enzyme-substrate complex, the Ser102 becomes fully deprotonated for nucleophilic attack and there is an associated transfer of this proton to the Mg- coordinated hydroxide group to form a Mg-coordinated water molecule. Coordination of Zn2 stabilizes the deprotonated Ser102. 31 - O-(Ser102) (Mg2+)-OH 2 (Zn2+)

32. 32 Mechanism: The Three Metal Triad In the first in-line displacement, the negatively charged Oxygen of Ser102 attacks the phosphorus center of the substrate in the enzyme-substrate complex to form a covalent serine-phosphate intermediate Zn1 participates in this step by coordinating the bridging oxygen atom of the substrate and facilitating the departure of the alcohol leaving group. 32 O-(Ser102) P OR O (Zn2+)

33. 33 Mechanism: The Three Metal Triad In the second in-line displacement step, a nucleophilic hydroxide ion coordinated to Zn1 attacks the phosphorus atom, hydrolyzing the covalent serine-phosphate intermediate to form the non-covalent enzyme-phosphate product complex and regenerate the nucleophilic Ser102. Zn1 lowers the pKa of the coordinated water molecule to effectively form the nucleophilic hydroxide ion 33 - O-(Ser102) Pi

34. 34 Mechanism: The Three Metal Triad The Mg-coordinated water molecule acts as a general acid to reprotonate the oxygen of Ser102. Protonation of Ser102 may facilitate departure of the phosphate product from the non-covalent complex. 34 HO-(Ser102) Product (Mg2+)- - OH

35. 35 Mechanism: The Three Metal Triad Alternatively, the Mg-coordinated water molecule may directly protonate the phosphate group for its release. The release of phosphate from the complex to give the free enzyme may be facilitated also by the increased mobility of the Arg166 side-chain. 35

36. 36 -Mechanism: Three Metal Triad Stec et al. 1309

37. Conclusion

38. Co-factors were successfully removed from Alkaline phosphatase shown by inactivity post EDTA Addition of Co-factors were successfully reintroduced and activity was partially restored. It was found that Mg alone did not restore AP activity, but did help increase activity in the presence of Zn. Zn alone can restore activity but is more effective in the presence of Mg.

39. Application

40. The Importance of Zinc in biological systems Phosphorylation is important in process in biological systems. As seen by the data from this experiment, the concentration of Zinc in the environment of the Alkaline Phosphatase is essential for activity. Numerous experiments have been done on AP. As Garen and Levinthal state, AP can be studied in a number of conditions. Zinc concentration is another condition that can be monitored.

41. 41 Phospholipase C (PLC) This three metal-ion mechanism proposed for alkaline phosphatase can be related directly to a whole class of enzyme mechanisms involving three metal ions, such as that observed in phospholipase C Enzymes which cleave phospholipids just before the phosphate group Synonymous with the human forms of this enzyme, which play an important role in eukaryotic cell physiology: signal transduction pathways. 41 http://en.wikipedia.org/wiki/File:Phospholi pases2.png

42. Further Experiments Complete inactivation of enzyme was not achieved. For future studies observations at complete enzyme inactivation would result in more concrete results. The role of pH in the enzyme catalysis is of particular interest for further investigation. Because alkaline phosphatase requires a higher pH for optimal activity the removal of metals at different pH and re addition could provide interesting results on the mechanism of AP. When Zinc is reintroduced to AP, oxygen my be deprotonated which would lower the pH of the environment and affect the protein activity because of the acidic interactions in other areas. Proteins stability is dependent on structure. Magnesium provides structural stability, so addition of cofactors at different temperatures could result in interesting results. Different metal ions could be analyzed for its affect on the metal binding sites. For example, the affect of Co2+

43. References

44. Coleman, J. (1992) “Structure and Mechanism of Alkaline Phosphatase” Annu. Rev. Biophys. Biomol. Struct. Garen, A., and Levinthal, C. (1959) “A Fine-Structure Genetic and Chemical Study of the Enzyme Alkaline Phosphatase of E. coli.” Biochimica et Biophysica Acta. Ninfa, A., Ballou, D., and Benore, M. (2010) Basic Procedures in the Biochemistry Laboratory, in Fundamental Laboratory Approaches for Biochemistry and Biotechnology. 2 nd Ed. Stec, B., Holts, K., and Kantrowitz, E. (2000) “A Revised Mechaniem for the Alkaline Phosphatase Reaction Involving Three Metal Ions.” J. Mol. Biol. Zalatan, J., Fenn, T., and Herschlag, D. (2008) “Comparative Enzymology in the Alkaline Phosphatase Superfamily to Determine the Catalytic Role of an Active-Site Metal Ion.” J. Mol. Biol.

45. 45 References Hough, E., Hansen, L. K., Birknes, B., Jynge, K., Hansen, S., Hordvik, A., Little, C., Dodson, E. J. & Derewenda, Z. (1989). High-resolution (1.5 AÊ ) crys- tal structure of phospholipase C from Bacillus cereus. Nature, 38, 357-360. http://en.wikipedia.org/wiki/Phospholipase_C 45