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Phosphorus - the Giver of Life

G. Michael Blackburn

Krebs Sheffield Institute University

Krebs Sheffield Institute University

Department of Molecular Biology and Biotechnology

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3HIGHI*

EDINBURGH

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“What  really  interests  me  is  whether  God  could  have  created  the  world  any  differently;  in  other  words,  whether  the  demand  for  logical  simplicity  leaves  any  freedom  at  all.”                  Albert  Einstein  

“Where  there’s  life,  there’s  phosphorus”  Sir  Alexander  Todd,  Kyoto  Lecture,  1982    

         Life  on  earth  depends  on  Phosphorus:  uniquely!  

1.  The  variety  of  roles  of  phosphate  in  life:    are  they  endless?  2.  How  did  phosphorus  get  to  earth?    Are  we  a  lucky  Planet?  3.  Morocco’s  centrality  for  phosphate  and  its  global    future  –  sustainable  or  not?  

4.  The  paradox:    extreme  structural  stability    vs.  funcOonal  reacOvity  5.  Is  there  any  alternaOve  to  phosphate?  6.  How  has  protein  crystallography  resolved  this  central  paradox  of  life?  

Molecular  Issues  

Global  Issues  

Rabat  02  

Rabat 03"

1. Multiple Major Roles for Phosphate in Life!The RNA World - life likely started with RNA preceding DNA and #

"proteins - for both genome and catalysts !

Genome stability - DNA structure demands ultra-stable phosphate #"diesters - and repair processes on demand for survival!

Lipids - cell membranes utilise phospholipids as essential stable components! Skeletal structure - calcium phosphate (apatite) #

"as a key mineral component! Compartmentalisation – ionised phosphate esters are #

"unable to cross membranes and can be trafficked! Energetics - Energy-rich phosphates especially Adenosine Triphosphate [ATP]"

Organelle membrane recognition - phosphoinositols!

Signalling – most catalytic proteins become phosphorylated# by Kinases to modulate their activity and second messengers, #

cAMP and cGMP, are vital for cell signalling!

GTP hydrolysis is the core of cell signalling in man (30 s-1)"

ATP!

Energetics - ATP

RO!R O! P!

O!!

O–!

Phosphate"esters!

CML drug!

Gleevec!

2. Galactic & Earth Origin of Phosphorus!•! CI$*.8B9%,7$<%@/+BEGO+8%+?%S.%"8B%T70%"9%

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16O + 16O ––> 31P + p + 7.7 MeV

16O + 16O ––> 28Si + 4He + 9.6 MeV

16O + 16O ––> 31S + n + 1.5 MeV

16O + 16O ––> 30P + 2H - 2.4 MeV

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‘Life can multiply until all the phosphorus is gone, and then there is an inexorable halt which nothing can prevent. We may be able to substitute nuclear power for coal, and plastics for wood, and yeast for meat, and friendliness for isolation - but for phosphorus there is neither substitute nor replacement’. Q9""G%597N+=%g]7?.K9%*+^-.8.GHh%'i24%

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Rabat 08"

4.   Does Stability of its Mono- and Di-Esters make it Unique?

5.   Is there an Alternative Element to Phosphorus for Life?

6.   How has Evolution achieved the necessary Dynamics for its

Multiple uses of Phosphate Esters?

Part  2.      How  did  Evolu'on  select  Phosphorus    for  its  Mul'ple  Roles  in  Life?  

4. The Central Paradox of Phosphate"Esters: Stability vs Mobility!

Triose phosphate!isomerisation! 2 days"

CO2 hydration" 5 sec"

(RO)2PO2– hydrolysis !

108 years"

Peptide hydrolysis! 450 years"

Uncatalysed reactions!t1/2 in water at 25 ˚C"

MeOPO3= hydrolysis !

1012 years"

Hence DNA stability!

Lad, C; Williams, N.H.; Wolfenden, R.; PNAS, 2003, 100, 5607-10"

How good are Enzymes for the Hydrolysis of Phosphate Esters? Enzymatic Hydrolysis of Phosphate

Mono-esters in Water at 37 ˚C

Enzyme Substrate kcat s-1 kcat/Km M-1s-1

PPI Phosphorylase Pase-" 39 4 x 106 FBPase Fructose 1,6-BP 21 1.5 x107 IP Inositol 1-P 22 3 x 105 No Enz Methyl Phosphate 2 x 10-20 (kuncat)

Thus, kcat/kuncat = 1021

Rabat 09"

S^H*-^H*

•  Dialkyl sulfates: non-ionic, excellent alkylating agents, totally unfitted for a structural role in biology"

•  Monoalkyl sulfates: anionic, used in biology for compartmentalization, lipid structure, signaling etc. BUT too high energy of formation ( it takes 2 ATP to make one sulfate ester). " " "(Richard Wolfenden & Yang Yuan, PNAS, 2006). "

•  Similarities between arsenic acid and phosphoric acid raise the possibility that arsenate can replace the phosphate in biological molecules such as the hexose phosphates and adenosine triphosphate. Condensed arsenates such as K4As2O7 are known in the solid state. BUT, the As–O–As in these compounds is extremely unstable in water. Thus, species containing an arsenic-oxygen-arsenic group cannot be present in aqueous media. (Westheimer, Science 1987). "

•  Neutral esters of arsenic acid such triorganyl arsenate can only be made in non-aqueous conditions. The As–O–C bond is unstable in water. "

•  Mono- and diesters of arsenic acid have never been isolated and arsenic acid esters are very much more easily hydrolyzed than phosphoric acid esters.

•  Silicic acid is more abundant in nature than phosphoric acid and is tetravalent. It is non-viable because Its esters hydrolyze far too rapidly to survive. Silicic acid is also too weak an acid: pKa1 ~ 9.5. Moreover, its diesters cannot ionize in neutral solution to carry a negative charge. "

Rabat 10"

4. Stability - There is No Alternative to Phosphate Di- and Mono-Esters!Element   Oxyacid   Crust  

Abundance   pKa1   pKa2   pKa3   Diester  t1/2  y  

Diester  charge  

Bond  cleaved  

Monoester    t½  y  

Monoester  charge  

Bond  cleaved  

P   H3PO4   Low   2.1   7.2   13.1   108  y   1  -­‐ve   P–O   1012  y   2  –ve   P–O  

S   H2SO4   Medium   >0   2.0   –   1.7  h   0   C-­‐O   1100  y   1  –ve   C–O  

As   H3AsO4   Medium   2.2   7.0   11.5   <2min   1  –ve   As–O   6  min   2  –ve   As–O  

Si   H4SiO4   High   9.5   >13   –   <1min   0   Si–O   <  1  min   0   –  

6. What makes Phosphate Esters so Stable?!

Frank Westheimer’s more perceptive line!“Phosphoric acid is specially adapted for its role in nucleic acids because it can link two nucleotides and still ionize; the resulting negative charge serves both to stabilize the diesters against hydrolysis and to retain the molecules within a lipid membrane…. Phosphates with multiple negative#charges can react by way of the #monomeric metaphosphate ion, #PO3

–, as an intermediate. "No other residue appears to fulfil the #multiple roles of phosphate in biochemistry.”

F. H. Westheimer, Science (Washington, DC, US) 1987, 235, 1173.

Adenosine 5 -triphosphate

Adenosine 5 -phosphate

The Anionic Shield !

ATP + AMP ADP + ADP!

Adenylate Kinase has to #overcome anionic repulsion of #

4 -ve vs 2 -ve charges. Yet it achieves an acceleration of 1012"

!Alexander Todd s appraisal of the suitability of phosphate!“Let’s try to summarise what Nature needs to facilitate all she has to do with #

carbon-based building bricks."1)! She needs a strong acid capable of forming anhydrides which can be used#

for energy storage and transport. "2)! The acid must be tribasic so that it may act as a link between two molecules of #

groups and still have one free acidic hydroxyl for further reaction."3)! The strength of the acid is important since it permits use in carbon-carbon bond synthesis.!

Kyoto lecture 1981

Rabat 11

groups and still have one free acidic hydroxyl for further reaction.

monomeric metaphosphate ionmetaphosphate ionmetaphosphate ,PO3

–, as an intermediate.

Rabat 12

6. How has Evolution achieved the Necessary Dynamics?

1.  Optimize Geometry – “In-Line” Phosphoryl Transfer

2.  Charge Balance – Neutralize the “Anionic Shield”

3.  Desolvation – Activate the nucleophile and the electrophile

4.  Bring the Reactants close together – “Associative Mechanism”

Five  Fundamentals  in  Catalysis  of  Phosphoryl  Group  Transfer  (PO3)–  

Phosphoryl  Group  

6.1 In-Line Attack of Nucleophile is Universal in Biology

Rabat 13

In the natural state, A = B = C = oxygen-16, so the phosphorus atom is Pro-Pro-chiral. Make the phosphorus chiral and study the stereochemistry of the reaction: Make A = oxygen-18, B = oxygen-17, C = oxygen-16 (Knowles, Lowe, others)

RESULTS: Inverting Enzymes (Hexokinase, Glycerol kinase, all kinases) use one-step phosphoryl transfer

Retaining Enzymes : [Phosphoglycerate mutase (histidine) Alkaline phosphatase (serine) !-Phosphoglucomutase (aspartate) Tyrosine phosphatase (cysteine)] use two-step phosphoryl transfer.

NB Retention of configuration at phosphorus always means a 2-step reaction, both steps being inversion

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6.2 In-Line Attack is manifest in Protein Crystallography

Rabat 14

RhoA.RhoGAP-GDP-MgF3– !

D.M.Graham, Chem. Biol., 2002, 9, 375-81!

UvrD-DNA-ADP-MgF3–!

J.Y.Lee, W.Yang, Cell, 2006 127, 1349-60 !

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M.J.Cliff, M.W.Bowler et al., JACS, 2010, 132, 6507-16!

6.4 Multiple In-Line Attack in Metal Fluoride Complexes

Rabat 15

Isosteric!Isoelectronic!

Analog!

111 Examples in the PDB. All tetrahedral "mimics of stable phosphate dianion (1995-date).!

Myosin.ADP.BeF3!Rayment I. et al., Biochemistry 1995, 2.0Å !

Beryllium Trifluoride Anion, BeF3–!

102 Examples in the PDB. All octahedral with 2 apical oxygen ligands. (1995 - date)!

RhoA-GTPase "Rittinger K, et al., Nature 1997, 1.68 Å !

Tetrafluoroaluminate Anion, AlF4–!

52 Examples in the PDB. All trigonal bipyramid with 2 apical O or N ligands (1997 - date).!

NDP Kinase!Cherfils et al., PNAS 1997, 2.0Å !

Aluminum Trifluoride, AlF30!

16 Examples in the PDB. All tbp with 2 apical oxygen ligands (2002 - date).!

RhoA-GTPase "Graham et al., Chem.Biol. 2002, 1.80 Å !

Trifluoromagnesate Anion, MgF3–!

6.4!

7.2!

8.0!

8.8!

9.2!

pH!

0.5 mM PSP, containing 10 mM MgCl2, 3 mM Al3+, 6 mM NH4F, 2 mM TCEP, (No Serine)

FB FD FC FA AlFx

FA FC FB

The AlF30 Structure initially assigned

for phosphoserine phosphatase is, in fact, MgF3

–

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TRANSITION STATE STUDIES ON ENZYMATIC PHOSPHORYL TRANSFER

6.5. Charge Balance is used to overcome the Anionic Shield

Adenosine 5 -triphosphate

Adenosine 5 -phosphate

The Anionic Shield !

ATP + AMP ADP + ADP!

Adenylate Kinase has to "overcome anionic repulsion of "

4 -ve vs 2 -ve charges. Yet it "achieves a rate acceleration of 1012!

4 -ve

2 -ve !!!!

"#

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Charge Balance sums the Pauling Atomic charges inside a sphere of radius r centered on phosphorus

Kinases

Rabat 18

Relative positions of ADP and 1,3-Bisphosphoglycerate In Phosphoglycerate Kinase.

The Energy to bring them together from infinity is ca. 40 kcal/mol (DFT calculation) Matt Bowler (Grenoble), Nigel Richards (Florida)

ADP 3-ve ADP 3-ve ADP 3-ve

4-ve 1,3-BPG

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Rabat 19

Association and Dissociation described in terms of (a) Bond Lengths or (b) Bond Orders. !As the connection is an exponential function (Linus Pauling) they offer very different interpretations of the same situation.!

In Bond Length terms: in a Dissociative process (upper) the AO--P bond substantially lengthens before the P—OB bond forms; A Concerted process has bond making synchronous with bond breaking. In an Associative process (lower) there is P-OB bond forming in advance of AO—P bond breaking.

6.8. Bring the Reactants close together – “Associative Mechanism” quasi metaphosphate

The question is: “How to Bring the Reactants close together – against the anionic shield?”

Jin,  Yi,  et  al.  PNAS,  111  (2014),  12384-­‐12389.  

Rabat 22

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Rabat 22

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CONCLUSIONS!

1.   The unique properties of Phosphate are:(a) !Highly stable diesters(b) !Ultra-stable monoesters(c) !Water solubility(d) Anionic character for mono-and di-esters(e) !Acceptable energy of formation of R–O–P bonds(f) !Significant release of energy on hydrolysis of P–O–P bonds(g) !Adequate elemental availability throughout the Universe through SNe!

2.   No other element has esters of its oxyacids with the required stability, anionic character, and water-solubility to support life / and low pKa. Sulfates, arsenates, and silicates all fail to meet these requirements. !

3.   In magnesium, phosphate anions have found the perfect complement to modulate their behaviour.!

4.   Evolution has found a surprisingly easy route to catalyse phosphoryl group transfer – by overcoming the “Anionic Shield” with complementary charge in the transition state. !

5.   Protein crystallography has revealed ground state, transition state analogue, and product complexes for phosphoryl transfer reactions that have enormously advanced our understanding of how enzymes work.!

6.   It thus seems inevitable that Wherever there is Life in Our Universe, there will be Phosphate; its esters and anhydrides fulfilling comparable roles in structure, signalling, and energetics.!

Rabat 26

Debabrata Bhattasali (Dalhousie)!Nicky Baxter ! !!David Berkowitz (Nebraska)!Wolfgang Bermel (Bruker)!Matt Bowler (EMBL)!Matt Cliff (Manchester)!Jo Griffin ! !!Andrea Hounslow!!!

David Jakeman (Dalhousie)!Yi Jin (York)!Xiao-Xia Liu (Dallas) !Erika Pellegrini ! (EMBL) ! !!Jon Waltho ! !!Edwin Webster (Memphis)!Mike Williamson !!

All Thanks Go To

V#=#4(M>(

David Jakeman (Dalhousie