1. Neemah BassiriRad Jennifer Bao BIOC 463a Spring 2012
Effect of Zn2+ Metal Chelation on Catalytic Activity of Alkaline Phosphatase
Neemah BassiriRad
Jennifer Bao
BIOC 463a Spring 2012

2. R-OPO3H- + H2O  R-OH + H2PO4-
Introduction
Alkaline phosphatase (AP) is an enzyme widely studied for its prevalence in mammalian and bacterial organisms.
Catalyzes the hydrolysis of phosphate monoesters, with maximum activity occurring in environments of pH greater than 7.0:
R-OPO3H- + H2O  R-OH + H2PO4-

3. Mechanism
Zn2+ and Mg2+ are proposed to be important for catalytic activity:
OH- coordinated by Mg2+ deprotonates Ser102
Zn2+ stablizes alkoxide of Ser102
Covalent intermediate formed when Ser102 attacks the phosphoryl phosphate atom
Zn2+ ion nearby stabilizes intermediate and promotes the liberation of RO-
OH- coordinated by Zn2+ ion mediates the liberation of Pi and regeneration of Ser102

4. DTPA: A Chelator of Metals
Zn2+-specific chelating agent

5. Hypothesis
Addition of DTPA to AP will result in loss of activity via chelation of Zn2+ ions from the active site.
Loss of Zn2+ ions will be verified through inductively coupled plasma mass spectrometry (ICP-MS).

6. Methods: ICP-MS
Provides high-resolution measurements of a broad range of metals
Samples are atomized and ionized with a plasma torch at 10,000 K.
Coupled mass spectrometer then separates and detects the ions.
Sensitivity of extends to parts per billion (ppb) precision.
More resolved than atomic absorption methods of metal detection, such as flame atomization
Samples are not ionized as completely as compared to a plasma torch.

7. Experimental Design
Initial dialysis of AP from Sigma-Aldrich to remove contaminants
Chelation of Zn2+ was carried out using ten concentrations of DTPA
0.5 mL samples
5 μM AP per sample
[DTPA] = 0 – 500 μM
Activity assays were performed after one hour of incubation at pH 8 and room temperature ([S] >> Km).
Submitted three samples to ICP-MS:
[DTPA] = 0 μM
[DTPA] = 500 μM
10 mM Tris buffer pH 7.4

8. Results

9. Results
62.86% of Vmax was lost when 50 equivalents of DTPA were added.
62.97% of Zn2+ was lost when 50 equivalents of DTPA were added.
Sample
[DTPA] μM
V0 (μmol PNPO-/min) 1
183.4 2 5
159.2 3 10
125 4 15
120.1 20
116.7 6 25
120.3 7
90.4 8
250
85.2 9
375
74.7
500
68.1

10. Conclusions
DTPA effectively reduced AP catalytic activity by removal of Zn2+
Removal of Zn2+ was confirmed with ICP

11. References
Ninfa, Alexander J., David P. Ballou, and Marilee Benore. Parsons. Fundamental Laboratory Approaches for Biochemistry and Biotechnology: Alexander J. Ninfa, David P. Ballou, Marilee Benore. Hoboken, NJ: Wiley, 2010.
Stec, B., Holtz, K.M., and Kantrowitz, E.R. (2000) Journal of Molecular Biology 299,