Purple acid phosphatase (PAP) is an enzyme containing heterovalent iron and zinc ions at its active site that catalyzes the hydrolysis of phosphomonoesters. It is present in both plants and animals, playing roles in bone resorption and phosphate acquisition. Several small molecule models of the PAP active site have been synthesized containing dinuclear iron or mixed iron-zinc centers that accurately mimic the electronic structure and catalytic activity of the enzyme. Kinetic studies on these models provide insight into the reaction mechanism of PAP-catalyzed hydrolysis.

1. PURPLE ACID PHOSPHATASE
Rohit Samanta
2018MSCH024

2. INTRODUCTION
• Purple acid phosphatase(PAPs) belong to the family of dinuclear
metallohydrolases.
• Purple acid phosphatase(PAPs) catalyzes the hydrolyses a wide range of
phosphomonoester and amide substrates.It is present both in animals
and plants.
• This enzyme have many roles especially in animals(BONE RESORPTION)
and phosphate acquisition in plants.
• The active site of this enzyme contains heterovalent(Fe3+ and an M2+ )
where M can be iron, zinc, manganese.
• Mamalian PAPs are bifunctional catalysing both hydrolytic reactions and
peroxidations. The later are facilitated by the redox active iron and
mechanism goes by Haber-Weiss-Fenton equations.

3. STRUCTURE
Plant PAP

4. DESCRIPTION OF STRUCTURE
• Different PAPs have different active site structure. Plants have mainly
enzymes of 55kDa while mammals have enzymes of 35 kDa
monomers.
• M(III) is stabilised by (one aspartate, one tyrosine and one histidine)
and M(II) is stabilised by (two histidines and one asparagine) and for
bridging one hydroxo and one aspartate residue is present.
• The characteristics purple colour of this enzyme is due to charge
transfer transition from a conserved tyrosine ligand to ferric ion in the
active site(wavelength=510-560 nm molar extinction constant=3000-
4000 M-1cm-1 .
Coordination Chemistry Reviews 257 (2013) 473– 482

5. • a terminal hydroxo ligand coordinated to iron (Fe-O 1.9 A), a terminal
aqua ligand coordinated to zinc (Zn-O 2.2 A), and a p.-hydroxo bridge
(Fe-O 1.9 A, Zn-O 2.1 A). The two terminal solvent ligands are about
2.8 A apart in this model and therefore within hydrogen bonding
distance. Whereas the zinc coordination sphere matches a distorted
octahedron, the symmetry of the iron ligand sphere is almost
perfectly octahedral with respect to the angles around the metal .
• The bimetal active centre of the enzyme is generally
antiferromagnetically coupled.

6. CATALYTIC CYCLE
Coordination chemistry reviews 257(2013)

7. Importance of PAP
• The physiological roles of mammalian PAP have been associated with
are
• Iron transport,generation of reactive oxygen species(ROS) as an
immune response, increased bone resorption.
• In plant PAP the major function is scavenging of inorganic phosphate
from organophosphate in soil. In plants also due to pathogen attack
extracellular increase in ROS is observed.

8. Coordination Chemistry Reviews 257 (2013) 473– 482

9. Mimicing active site structure
• Model 1:-
2-{[bis(2-methylpyridyl)-amino]methyl}-6-{[(1-methylimidazole-2-yl)methyl)(2-pyridylmethyl)amino]methyl}-
4-t-butylphenol
ACS Omega 2017, 2, 4737−4750

10. • Ligand to metal charge transfer(LMCT) transition appearing at 518-
559nm(native enzyme 510-560nm). [Tyrosine-Fe(III)].
• M-M distance in these binuclear enzyme attributes to the observed
antiferromagnetic coupling between the two metal centre. The
slightly longer M-M distance[3.404-3.431 Å ] in the complexes
prepared can be attributed due to presence of acetate bridging
moieties present between the metal centre compared to one
hydroxide moiety bridging two metal centre in enzyme active site.

12. • In a mechanistic reaction with (2,4-BDNPP) the pH of the reaction should be
around 7 because in alkaline medium PAPs doesn’t show any enzyme activity.
• In the equilibrium this species contain a labile water which gets replaced by (2,4-
BDNPP) after which nucleophilic attack occurs from Fe(III). Due to high pH
deprotonation occurs thereby inhibiting the 2,4-BDNPP coordination to metal
centre.
• Excellent result is that hydrolysis of 2,4-BDNPP is 5900 times faster than the
uncatalyzed reaction.

13. •
hydrolysis of 2,4-BDNPP using four different catalyst prepared
This data shows that the pka3 which is
responsible for depronation of M(II)
coordinated water is around pH=7 which is
same for enzyme.

14. MECHANISM OF HYDROLYSIS OF 2,4-BDNPP
3a=Zn
3b=Ni
4a=Co
4b=Cu

15. KINETICS EXPERIMENT

16. Model system
2-{[bis-(pyridin-2-ylmethyl)amino]methyl}-6-{[2-
hydroxybenzyl)-(pyridin-2-ylmethyl)amino]methyl}-4-
methylphenol
Inorg. Chem. 2015, 54, 7249−7263

17. • The generation of Fe(iii)Fe(ii) complex of H3L4 was expected to create an accurate structural, spectroscopic
and functional model for PAP. For oxidation from Fe(ii)Fe(ii) to Fe(iii)Fe(ii) is done by ferrocenium
hexaflourophosphate.
• EPR spectroscopy:
Inorg. Chem. 2015, 54, 7249−7263

18. Kinetics experiment
Under basic conditions the asymmetric diiron complex
shows higher TONs than the symmetric isomer
The TON of the asymmetric diiron complex
(H2L4) is dependent on the oxidation state of
iron measurement at pH at 9 and 9.5 resulted in
a higher TON. However in strong base the trend
is reversed Fe(iii)Fe(ii) complex reveals higher
TON.

19. (L = 4,11-di-methyl-1,8-bis[N-(2-di-(2-pyridyl)-
methyl)ethylamino]cyclam) ligand
Coordination Chemistry Reviews 257
(2013) 473– 482

20. CONCLUSION
• the heterovalent diiron complex [FeIIIFeII(H2L4)(solv)x]4+, obtained by
oxidation of [FeII2(H2L4)(μ-OAc)2]+ with ferrocenium hexafluorophosphate
under anaerobic conditions, could be trapped and characterized by 57Fe
Mössbauer, UV-vis, and EPR spectroscopy. [FeIIIFeII(H2L4)(solv)x]4+ is an
accurate structural model for the active form of mammalian PAPs with a
heterovalent diiron core in an asymmetric geometry and, since it is phosphatase
catalyst, it also is a functional model.