Lecture 9: Metals & EnzymesOct. 24th

Role of metals in basic geobiochemical cycles

Morel & Price Science (2003) 300:944

N cycle

Metalloproteins • in vivo metal concentrations (E. coli)

– K, Mg 108 atoms/cell ~10 mM– Ca, Zn, Fe 105 atoms/cell ~ 0.1 mM– Cu, Mn, Mo 104 atoms/cell ~ 10 µM– V, Co, Ni low abundance

• Estimated ~1/3 of all proteins contain metals– Na, Mg, K, Ca– V, Mn, Fe, Co, Ni, Cu, Zn– Mo, Cd, W

• Metalloproteomics:– Structural and functional annotation of

proteins in structural genomics– Shi et al Structure (2005)13:1473

• 10-15% contain stoichiometric amounts of transition metals

Finney & O’Halloran Science (2003) 300:931

Proteinligands• Majorligands

– Cys,His,Asp,Glu• Rarerexamples

– Met (cytochromec,azurin)– Tyr (dioxygenase,catalase)– Asn (Cabindingproteins,lipoxygenase)– Gln (subBlisinCa,stellacyanin?)– Ser (MoFe-protein,ferredoxins,DMSOreductase)– Lys (phosphoenolpyruvatecarboxykinase)– carbonylO (Ca2+sites)– amideN (Pcluster/nitrilehydratase)– aminoN (cytochromef,CooA(pro))– carboxyC (lipoxygenase),– formylMet (MgofBchlinLH2)– Arg (bioBnsynthase)– Trp? noexamplesyet;Trpasradical-CytCperoxidase

Idealized coordination geometries

Typical metal-ligand distances for first row metals:M - O/N 2.0-2.1 ÅM - S 2.3 Å

His/Glu/Asp coord

Smallest coordination motifs 3 res - sq planar - A-cluster Carbon monoxide

dehydrogenase-aCoA (1mjg) 6 res - sq planar - Nitrile hydratase 14 res - Tetr- some Zn fingers 12 res - Oct - calcium sites

Harding Acta Cryst (2006) D62:678

HN

N

Nε2

Nδ1

HN

N

M

M

Hpreferredmonoprotonated

preferred in proteins (75%)

preferred in peptides

C

O

O

C

O

O

anti

syn

bidentate-1 -1

tetrahedral octahedral trigonal bipyramid square planar

distribution of Zn - N His in high resolution protein structures (res < 1.6 Å), relative to d = 2.00 Å observed in small molecule structures

Zn - O distances in PDB structures with bidentate carboxylate ligands(both d < 3.00 Å)

Distribution of metal ligand distances in protein structures

Harding Acta Cryst (2006) D62:678

Mg2+?

2.78

2.11

2.122.12,2.13 W2.39

2.29

Aldehyde ferredoxin oxidoreductaseChan et al Science (1995) 267:1463 (1aor)

Common components in protein solutions - often at high concIon M••O (Å) ion rad (Å) Favored coord num.Na+ 2.42 0.95 6Mg2+ 2.07 0.65 6K+ 2.84 1.33 7-8Ca2+ 2.39 0.99 6-8H2O ~2.8 4

Distinguishing Na+, Mg2+, K+, ( Ca2+) from H2O

?

Harding Acta Cryst (2006) D62:678

• Metal composition and quantitation:– ICP-MS (inductively coupled plasma - mass spectrometry)– atomic absorption– chemical methods

• Protein quantitation – protein - most difficult in practice - colorimetric methods– can be off by 100%+/-

• Nitrogenase– FeMo-cofactor - 1Mo:5-8Fe:6-9S (actual 1:7:9)

• Prismane protein with “6Fe:6S cluster” – Actually 4Fe:4S and 4Fe:3S:2O clusters

Characterization of metal sites: Stoichiometry

Howard & Rees Adv Prot Chem (1991) 42:199

Non-crystallographic methods for

characterizing metal centers

1.0 Å 1.0 Å1.3 Å1.0 Å1.3 Å2.0 Å

r = 2.0 Å

1.0Å1.3Å2.0Å3.0Å

dmax

Resolution dependence of electron density profiles…get negative ripples at ~resolution from scatterers

Crystallographic characterization of metal sites: resolution, accuracy elemental and oxidation state identity

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ρ r( ) = 4πs2 fFe s( )0

1 dmax

∫ sin2πsr2πsr

ds

Nitrogenase FeMo-cofactor 7Fe:1Mo:9S:homocitrate

Initial map3.5Å resolution

First “official” model2.8 Å resolution

2.2 Å resolution

Einsle et al Science (2002) 297:1696

6 Fe @ 2.0 Å and 9 S @ 3.3 Å from central ligand generate resolution dependent ripples at this site

FeMo-cofactor at 1.16Å resolution

A central light atom ligand (C)

Thelightatom!ItisCarbon!!

Spatzal..Rees&EinsleScience(2011)334:940 Lancaster..DeBeerScience(2011)334:974

X-rayemissionspectroscopy

Carbon

Nitrogen

Oxygen

Hintsatmechanism

Spatzal..Einsle,Howard&ReesScience(2014)345:1620 Anderson,Rifle&PetersNature(2013)501:84

Enzymes – biological catalysts• Not altered by reaction• Don’t change the

equilibrium (Keq)• Lower the activation

barrier• Unique

microenvironment (the active site)

• High specificity and efficiency

Catalyst Rate EnhancementPalladium (i.e.) 102-104

Enzyme >1020

E + S ⇋ ES ⇋ ES* ⇋ EP ⇋ E + P

Terminology• Typically ends in -ase• Established by IUBMB (1992)• EC numbers to classify enzymes

– EC 1 Oxidoreductases: catalyze oxidation/reduction– EC 2 Transferases: transfer a functional group– EC 3 Hydrolases: catalyze the hydrolysis of various bonds– EC 4 Lyases: cleave various bonds by means other than

hydrolysis and oxidation– EC 5 Isomerases: catalyze isomerization changes in a

molecule– EC 6 Ligases: join two molecules with covalent bonds

http://www.chem.qmul.ac.uk/iubmb/enzyme/

EC 1 Oxidoreductases

EC 1.1 Acting on the CH-OH group of donors EC 1.17 Acting on CH or CH2 groups

EC 1.2 Acting on the aldehyde or oxo of donors EC 1.18 Acting on iron-sulfur proteins as donors

EC 1.3 Acting on the CH-CH group of donors EC 1.19 Acting on reduced flavodoxin as donor

EC 1.4 Acting on the CH-NH2 group of donors EC 1.20 Acting on phosphorus or arsenic in donors

EC 1.5 Acting on the CH-NH group of donors EC 1.21 Acting on X-H and Y-H to form an X-Y bond

EC 1.6 Acting on NADH or NADPH EC 1.22 Acting on halogen in donors

EC 1.7 Acting on other nitrogenous compounds as donors EC 1.97 Other oxidoreductases

EC 1.8 Acting on a sulfur group of donors EC 2 Transferases

EC 1.9 Acting on a heme group of donors EC 2.1 Transferring one-carbon groups

EC 1.10 Acting on diphenols and related substances as donors

EC 2.2 Transferring aldehyde or ketonic groups

EC 1.11 Acting on a peroxide as acceptor EC 2.3 Acyltransferases

EC 1.12 Acting on hydrogen as donor EC 2.4 Glycosyltransferases

EC 1.13 Acting on single donors with incorporation of molecular oxygen (oxygenases)

EC 2.5 Transferring alkyl or aryl groups, other than methyl groups

EC 1.14 Acting on paired donors, with incorporation or reduction of molecular oxygen

EC 2.6 Transferring nitrogenous groups

EC 1.15 Acting on superoxide radicals as acceptor EC 2.7 Transferring phosphorus-containing groups

EC 1.16 Oxidising metal ions EC 2.8 Transferring sulfur-containing groups

http://www.genome.jp/dbget-bin/www_bget?ec:1.1.1.1

Example

Rate enhancement of enzymesEnzyme Uncatalyzed

rate (s-1)Catalyzed rate (s-1)

Rate enhancement

OMP decarboxylase 2.8 x 10-16 39 1.4 x 1017

Staphylococcus nuclease 1.7 x 10-13 95 5.6 x 1014

AMP nucleosidase 1.0 x 10-11 60 6.0 x 1012

Carboxypeptidase A 3.0 x 10-9 578 1.9 x 1011

Ketosteroid isomerase 1.7 x 10-7 66,000 3.9 x 1011

Triose phosphate isomerase 4.3 x 10-6 4,300 1.0 x 109

Chorismate mutase 2.6 x 10-5 50 1.9 x 106

Carbonic anhydrase 1.3 x 10-1 1,000,000 7.7 x 106

Cyclophilin 2.8 x 10-2 13,000 4.6 x 105

Radzicka & Wolfenden (1995) Science 267:90Rate enhancement is the catalyzed/uncatalyzed rates

Universeis1.4x1010

Reaction energetics• ΔG=ΔH-TΔS• ΔG=Gproducts-Greactants• ΔG < 0

– Proceeds forward– Exergonic (energy released)

• ΔG = 0 – At equlibrium– No net reaction

• ΔG > 0 – reaction goes in reverse– Endergonic (energy input)

• How do you get ΔG > 0 to move forward? Couple it to a ΔG < 0 reaction.

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vF = kF S[ ]eqvR = kR P[ ]eqvF = vR∴kF S[ ]eq = kR P[ ]eq

Keq =P[ ]eqS[ ]eq

=kFkR

S" P"kF"kR"

Transition state theory

Carbonic anhydrase

Fig. 5.1 & 5.2

ES ES‡ EI

CatalyzesthehydraBonofcarbondioxide

α(2CBB)

β(1I6P)

γ(1QRE)

Ribonucleotide reductase

Fig. 5.3 & 5.4

Tetrameric Class ITetrameric Class III

Dimer shown

Dimeric Class II Monomeric Class I

Catalytic subunits in blue

CatalyBcsubunit

ClassI&II

ClassIII

Two binding sites - multiple binding modes

Effector Substrate binding

dGTP dATP

dCTPDifferentbindingmodesoflooptoeffectacBvesite

Catalytic mechanism

Fig. 5.10

Free radical

Nordlund&Reinhard(2006)

Nucleotide hydrolases• Many different

nucleotide motifs– Gly-rich loop interacting

with P (Walker A/P-loop)– Aspartate to coordinate

Mg+2 (Walker B)• Needs a general base• Needs a positively

charged residue to stabilize build-up of negative charge

• Assembly of active site controls rate of NTP hydrolysis

• NTP binding sites often at subunit or domain interfacesCouples hydrolysis to conformational changes

Energetics• Concentrations

– [ATP] = 10mM– [ADP] = 0.1mM– [Pi] = 10mM

• ATP requirement– ~70kg person at rest

produces ~100 watts with a voltage drop of ~1.1V

– ~90 amps of current– ~2.6 ATP synthesized/

2e-

– ~50kg ATP synthesized daily

– ATP stores last 1-2 sec

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ATP→ADP + Pi

ΔG = ΔG°'+RT ln ADP[ ] Pi[ ]ATP[ ]

$

% &

'

( )

ΔG = −30 + 2.5ln 10−6

10−2$

% &

'

( )

= −30 − 23= −53kJ /mole

ΔG˚´isthestandardstatefreeenergychangeNeed~6kJ/moletogetanorderofmagnituderaBochange

Cellular nucleotide requirements• Metabolic reactions – mechanistic or energetic

requirements• Transcription/replication• Mechanical – transport, motility, unfolding, unwinding• Signaling

• In E. coli, ~56% of ATP utilized for protein synthesis• In nitrogen fixing organisms, ~40% of ATP for NH3

synthesis• In humans, substantial requirement for Na+/K+ ion

gradients

P-loop containing NTPases

SCOP classification (22 Families)http://scop.mrc-lmb.cam.ac.uk/scop/data/scop.b.d.fb.A.html

CATH: 3.40.50.300 (149 Families)

Myosin (actin) & Kinesin (MTs)

ATPases associated with a variety of activities

The glycine-rich loop• Nucleotide binding motif• P-loop or Walker-A• Examples

– GXXGXGK(S/T) – mononucleotide (Ras)

– GXGXXG – Dinucleotide (NAD in lactate dehydrogenase)

– GXGXXG – Protein kinase C– GXXG – Actin, hexokinase– GXXGXG – “GHKL”

domains – gyrases, histidine kinases

6q21 – Ras/GMPPCP

Ras(GTP)on

Ras(GDP)off

GDPGTPRas

Very unstable

0.02 min-1

GAPs 105X

GNEF/GEF

Ras has a slow off rate for GDP (~1.5 hr)

GDPGTP

Ras cycle

Effector

Effector*

30% of human tumors have altered versions of ras with mutations that retard GTP hydrolysis and leave in “on” state.

Ras-GTP vs Ras-GDP

Switch IT35 interacts

with Mg+2

Switch IIDXXGQ

D57 – Mg+2G60- γP

P-loop/Walker A6q21 GTP (Cyan)4q21 GDP (Green)

Switch loops sense γPiLeads to conformational changes

Mutations in G12 or Q61 most common in cancers

GAP – GTPase Activating Protein

• Used AlF – mimics a transition state?

• Ras-GAP-GDP-AlF3

• “arg finger” hypothesis

Scheffzek..Wittinghofer (1997) Science 277:333

Ras-RasGAP structure

1wq1Ras

p120 - GAP

Respiratory chain‘Chemiosmosis’ or ‘osmotic energy’

• Protons are pumped across the membrane by complexes– I NADH dehydrogenase– III (cytochrome bc1

complex)– IV (cytochrome c oxidase)– (II – succinate

dehydrogenase doesn’t pump)

• The gradient drives ATP synthesis

Fig. 5.11

PeterMitchellNobelChemistry1978(proposed1961)

ATP Synthase

Fig. 5.12 & 5.13

Structure of the F1 subunit

Fig. 5.14 & 5.15

1e79

α3β3γ

The asymmetric γ subunit has different contacts with the other subunits. This leads to conformational changes and differences in the nucleotide binding pocket in the β-subunits.

Abrahams et al. (1994) Nature 370:621

Model

• Model first proposed by Paul Boyer• In solution the reaction proceeds as shown• Synthesis is driven in reverse by the proton gradient

Fig. 5.17

Proof of principle

Fig. 5.18

Noji et al (1997) Nature 386:299

Active site

Fig. 5.19

Fo subunit

Fig. 5.20 & 5.22

Animatedmodel

GrahamJohnsonwww.fiVth.com