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

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-

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

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

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).

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.

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

Results

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

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

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, 1303-1311.