Enzymes. GENERAL FEATURES Enzymes are biological catalysts that speed up biochemical reactions.
Lecture 9: Metals & Enzymes Oct. 24thsaf.bio.caltech.edu/bi170/BMB170_2017_LECTURE9.pdf · Enzymes...
Transcript of Lecture 9: Metals & Enzymes Oct. 24thsaf.bio.caltech.edu/bi170/BMB170_2017_LECTURE9.pdf · Enzymes...
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Lecture 9: Metals & EnzymesOct. 24th
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Role of metals in basic geobiochemical cycles
Morel & Price Science (2003) 300:944
N cycle
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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
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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
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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
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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
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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
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• 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
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Howard & Rees Adv Prot Chem (1991) 42:199
Non-crystallographic methods for
characterizing metal centers
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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
€
ρ r( ) = 4πs2 fFe s( )0
1 dmax
∫ sin2πsr2πsr
ds
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Nitrogenase FeMo-cofactor 7Fe:1Mo:9S:homocitrate
Initial map3.5Å resolution
First “official” model2.8 Å resolution
2.2 Å resolution
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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)
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Thelightatom!ItisCarbon!!
Spatzal..Rees&EinsleScience(2011)334:940 Lancaster..DeBeerScience(2011)334:974
X-rayemissionspectroscopy
Carbon
Nitrogen
Oxygen
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Hintsatmechanism
Spatzal..Einsle,Howard&ReesScience(2014)345:1620 Anderson,Rifle&PetersNature(2013)501:84
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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
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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/
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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
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http://www.genome.jp/dbget-bin/www_bget?ec:1.1.1.1
Example
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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
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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"
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Transition state theory
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Carbonic anhydrase
Fig. 5.1 & 5.2
ES ES‡ EI
CatalyzesthehydraBonofcarbondioxide
α(2CBB)
β(1I6P)
γ(1QRE)
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Ribonucleotide reductase
Fig. 5.3 & 5.4
Tetrameric Class ITetrameric Class III
Dimer shown
Dimeric Class II Monomeric Class I
Catalytic subunits in blue
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CatalyBcsubunit
ClassI&II
ClassIII
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Two binding sites - multiple binding modes
Effector Substrate binding
dGTP dATP
dCTPDifferentbindingmodesoflooptoeffectacBvesite
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Catalytic mechanism
Fig. 5.10
Free radical
Nordlund&Reinhard(2006)
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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
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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
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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
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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
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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
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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.
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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
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GAP – GTPase Activating Protein
• Used AlF – mimics a transition state?
• Ras-GAP-GDP-AlF3
• “arg finger” hypothesis
Scheffzek..Wittinghofer (1997) Science 277:333
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Ras-RasGAP structure
1wq1Ras
p120 - GAP
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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)
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ATP Synthase
Fig. 5.12 & 5.13
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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
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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
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Proof of principle
Fig. 5.18
Noji et al (1997) Nature 386:299
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Active site
Fig. 5.19
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Fo subunit
Fig. 5.20 & 5.22
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Animatedmodel
GrahamJohnsonwww.fiVth.com