Determining the Zinc and Magnesium Dependencies of Alkaline Phosphatase

Andrew Ma, Elina Ly, Audrey Shi, and Ashley Vergara

BIOC 463aTuesday, November 22, 2011

Alkaline Phosphatase• Hydrolase enzyme found in E. coli• Located in the periplasm• Responsible for removing

phosphate groups from various chemicals

• Expression increases in the absence of phosphate

• Highly resistant to thermal inactivation and denaturation

• Exists as a dimer, each one containing two zinc and one magnesium ions

• Optimal activity at pH = 8.01. Stec, B., et al. (2000) “A revised mechanism for the alkaline phosphatase reaction involving three metal ions.” J. Mol. Biol., 299, 1303-1311.2. Garen, A. and Levinthal, C. (1959). “A Fine-Structure Genetic and Chemical Study of the Enzyme Alkaline Phosphatase of E. Coli.” Biochem.

Biophys Acta., 38, 483-494.

The Effects of Chelation• Chelating agents are chemical

compounds that bind certain metals by forming coordination bonds with the metal ions

• EDTA is a common chelator that forms 6 coordination bonds

• DTPA is a stronger chelator that forms up to 8 coordination bonds

• Chelating agents can remove metal ions from proteins and affect their overall activity

EDTA, a common chelator for many metals

DTPA, a stronger chelator for metal ions

Plock, D.J., and Vallee, B.L. (1962). “Interaction of Alkaline Phosphatase of E. coli with Metal Ions and Chelating Agents.” Biochemistry, 1, 1039-1043.

Experimental Purpose and Design• To determine how AP activity is affected when treated with the

chelating agent DTPA and how it compares to treatment with EDTA• Monitor effects by conducting activity assays and recording initial

velocity (Vo) readings for the following:*AP treated with EDTA-Incubation performed at room temperature-Varying incubation time

*AP treated with DTPA-Incubation performed at room temperature-Varying incubation time-Varying concentrations

Enzyme Preparation

• Dialyzed the AP with Tris buffer, pH = 7.4 to remove various metal-containing salts.• Dialysis was performed in a 50 kDA Amicon Centricon, which also concentrated the enzyme within a smaller volume

• Diluted 30 uL of purchased AP into 1 mL of Tris buffer, pH = 7.4.• The dilution was then added to a centricon.

AlkalinePhosphatase

Tris pH = 7.4

Assay Protocol

150 uL of 30 uM AP

15 uL of 0.1 M chelating agent

Incubate inactivation mixat room temperature

AP Inactivation Mix Kinetics Assay Mix

25 uL inactivation mix

450 uL Tris, pH = 8.0500 uL 1.2 mM PNPP

Read activity at 410 nm and record Vo

Results

Each curve represents the average of two inactivation assays.

0 5 10 15 20 25 300

1

2

3

4

5

6

7

f(x) = 5.90615 exp( − 0.0592292160231121 x )

f(x) = 5.08245 exp( − 0.122119350736687 x )f(x) = 5.31385 exp( − 0.0667097740277124 x )

10 mM EDTAExponential (10 mM EDTA)10 mM DTPAExponential (10 mM DTPA)5 mM DTPA

Time (min)

Vo (u

M/m

in)

5.68 min. 10.35 min. 11.75 min. Time at which activity is halved:

Conclusion

• Incubation with DTPA is an effective method for inactivating alkaline phosphatase activity

• DTPA is a chelating agent that inactivates AP nearly twice as quickly as does EDTA

• DTPA could possibly be used for other assays in which alkaline phosphatase activity needs to be inhibited

Future Directions

• Determining the pH, temperature, and concentration dependencies of AP inactivation via metal chelation

• Investigate whether only zinc ions are removed or if magnesium is also removed

• Observing AP’s activity when it uses other metal cofactors after treatment with DTPA

References• Coleman, J.E. (1992). “Structure and Mechanism of Alkaline Phosphatase.” Annu. Rev. Biophys. Biomol. Struct.,

21, 441-83. • Garen, A. and Levinthal, C. (1959). “A Fine-Structure Genetic and Chemical Study of the Enzyme Alkaline

Phosphatase of E. Coli.” Biochem. Biophys Acta., 38, 483-494.• Kim, E.E. and Wyckoff, H.W. (1991) “Reaction mechanism of alkaline phosphatase based on crystal structures.

Two-metal ion catalysis.” J. Mol. Biol., 218, 449-464.• Ninfa, Alexander J, Ballou, David P., and Marilee Benore. “Fundamental Laboratory Approaches for Biochemistry

and Biotechnology, Second Edition.” John Wiley & Sons, Inc.: 2010.• Plock, D.J., and Vallee, B.L. (1962). “Interaction of Alkaline Phosphatase of E. coli with Metal Ions and Chelating

Agents.” Biochemistry, 1, 1039-1043.• Stec, B, Holtz, K.M., and Kantrowitz, E.R. (2000). “A Revised Mechanism for the Alkaline Phosphatase Reaction

Involving Three Metal Ions.” J. Molecular Biology, 299, 1303-1311.