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SIMULACIONS COMPUTACIONALS PER A ENTENDRE COM FUNCIONEN ELS ENZIMS
Carme RoviraICREA― Universitat de Barcelona
Institut de Química Teòrica i Computacional (IQTCUB)
Què és un enzim?
Adaptat de https://www.ebi.ac.uk/training/online/course/introduction‐protein‐classification‐ebi/protein‐classification
• Els enzims són molècules essencials
• Els enzims són proteïnes
2016
Carme Rovira
Què és un enzim?
• Els enzims són proteïnes
• Els enzims actúen de catalitzadors per a accelerar les reaccions bioquímiques
• Els enzims no es modifiquen durant aquest procés
2016
Carme Rovira
Saliva: amilasa
Estómac:proteases
Budell prim:lipases,amilasa, lactasa
Dibuix dewww.divavillage.com
ENZIMS DIGESTIUS
Unitats de sucre(monosacàrids)
Molècula de carbohidrat
digestió
GLICOSIDASES 2016
Carme Rovira
Saliva: amilasa
Estómac:proteases
Budell prim:lipases,amilasa, lactasa
Dibuix dewww.divavillage.com
ENZIMS DIGESTIUS
Unitats de sucre(monosacàrids)
digestió
Enllaços glicosídics
GLICOSIDASES 2016
Carme Rovira
R. Wolfenden & M. J. Snider.Acc. Chem. Res. 2001, 34, 938‐945
The power of enzymes as catalysts
t1/2 = 1 billion years
t1/2 = 1 million years
t1/2 = 1 thousand years
t1/2 = 1 year
t1/2 = 1 day
t1/2 = 1 minute
age of the earth
O‐glycoside hydrolysis 5‐8 million years
2016
Carme Rovira
How enzymes work
PASS
AB
© 2010 Nature Education All rights reserved.
Without enzyme With enzyme
Energy
Reaction pathway
Activationenergy
A
B
A
B
Enzymes lower the activation energyof the biochemical reaction
2016
Carme Rovira
Enzymes are very specific
Example: α‐1,4‐glucosidase “cuts” glycosidic bondsbetween atoms 1 and 4 of the α‐glucose units of glycogen
n
1 4
...OH OH OH OH
2016
Carme Rovira
Lack of lactase causeslactose intolerance
GALACTOSE GLUCOSE
Enzymes and disease
Lack of α‐1,4‐glucosidase causes accumulation of glycogen in the liver, leading to the Pompe disease
GLYCOGEN
LACTOSE
GLUCOSE UNITS
2016
Carme Rovira
Knowing how enzymes work isimportant for drug design
Relenza Tamiflu
Synthesis of molecules that inhibit a disease‐related enzyme
2016
Carme Rovira
Sugars and common flu
Influenza virus
NeuraminidaseRelenza Tamiflu
N‐acetylneuraminic acid(sialic acid)
Approved since 1999
2016
Carme Rovira
Computer simulation to understandhow enzymes work
• Computer simulation provide molecular details of enzyme action
• Short‐lived states (e.g. transition states) can be identified
• Important for inhibitor (drug) design
Reaction pathway
A
B
2016
Carme Rovira
Impact of supercomputing
• The large increase of computer power in recent years has contributed enormously to the progress of computer simulation of enzymes (computational enzymology)
• Better models and precise methods can be used
• Meaningful predictions can be made
2016
Carme Rovira
Clarifying mechanisms of enzymesthat process carbohydrates
Gas2 glucosidase, an antifungal target
2016
Carme Rovira
Raich et al. J. Am. Chem. Soc. 138, 3325 (2016)
Clarifying mechanisms of enzymesthat process carbohydrates
2016
Carme Rovira
(Newton’s equations)
Molecular dynamics
Initial conditions:
• Atomic positions (e.g. crystal structure): {ri}• Atomic velocities (e.g. random velocities scaled to desired T): iv
Motion of atoms from
ri (t)i
2i
2
rr
dEd
tddmi
2016
Carme Rovira
Classical MD
E = Ebond + Eang + Edih + EvdW + Eelec
Energy from a force-field
I(r)ρ
Rρ(r),EminE Density Functional Theory
Molecular dynamics
Computer codes: GROMACS, NAMD, AMBER, …
Energy from quantum mechanics
Ab initio MD
Computer codes: CPMD, CP2K, QUANTUM EXPRESSO, …
C. RoviraMethods Mol. Biol. 305, 527 (2005)
Tobacco mosaic virusShulten & col.Structure 14, 437-449, 2006
2016
Carme Rovira
Classical MD
E = Ebond + Eang + Edih + EvdW + Eelec
Energy from a force-field
I(r)ρ
Rρ(r),EminE
Energy from quantum mechanics
Density Functional Theory
Molecular dynamics
Ab initio MD
2016
Carme Rovira
Active center
(quantum mechanics)Rest of the system
(molecular mechanics)
Multiscale methods
“for thedevelopment of multiscale modelsfor complexchemical systems”
The quantum mechanics / molecular mechanics (QM/MM) approach
ab initio MD classical MD
2016Carme Rovira
Carbohydrate degradation by glycosidases
glycosidic bondcleavage
sugar units(monosaccharides)
2016Carme Rovira
Carbohydrates are very flexible
Slide from Lluís Raich(University of Barcelona)
Sugar shapes
Chair
Most abundantfor a free sugar
Envelope Boat
2016
Carme Rovira
Biarnés et al. J. Biol. Chem. 2006 Biarnés et al. J. Am. Chem. Soc. 2011
Sugar distortion facilitates thebiochemical reaction
Sugars change shape when they bindto enzymes
twistedboat
O
Collaboration withAntoni Planas(IQS‐URL)
2016
Carme Rovira
Biarnés et al. J. Biol. Chem. 2006 Biarnés et al. J. Am. Chem. Soc. 2011
Sugars change shape when they bindto enzymes
Sugar distortion facilitates thebiochemical reaction
Energy
Reaction pathway
ActivationenergyA
B
2016
Carme Rovira
http://biology.tutorvista.com
Example: Golgi α‐mannosidase II
Golgi complex
Cell
mannose2
Anticancer target
2016
Carme Rovira
Mechanism of action
Golgi α‐mannosidase II
Petersen et al. J. Am. Chem. Soc. 132, 8291 (2010)
boat
Initialcomplex
O
Collaboration withPeter Reilly(Iowa State University)
2016
Carme Rovira
Petersen et al. J. Am. Chem. Soc. 132, 8291 (2010)Williams et al. Angew. Chem. Int. Ed. 53, 1087 (2014)
Golgi α‐mannosidase IIMechanism of action
anticancer inhibitors
transitionstate
2016
Carme Rovira
Privateer software: conformational validation of carbohydrate structures Agirre, Iglesias‐Fernández, Rovira, Davies, Wilson, Cowtan. Nat. Struct. Mol. Biol. 22, 833 (2015)
Computer simulation for structural validation
wrong conformation correct conformation
(high energy)
β α
(low energy)
Collaboration withGideon J. Davies(Universityof York)
Jon Agirre(Universityof York)
2016
© 2010 Nature Education All rights reserved. http://commonfund.nih.gov/Singlecell/index
Sugars on the cell surface
cytoplasm
cell surface carbohydrates (glycans)
2016
Carme Rovira
Outsideof cell
Cellmembrane
Insideof cell
(cytoplasm)
virusbacteria
Sugars on the cell surface
Special Issue.March 2001: Vol. 291. no. 5512.
Carbohydrates and Glycobiology
2016
Carme Rovira
Problems associated to a failure of cell‐cell communication
Outsideof cell
Cellmembrane
Insideof cell
(cytoplasm)
virusbacteria
Inflammation
Autoinmune
diseases:
asthma
diabetes
allergies
multiple sclerosis
Infection
2016
Carme Rovira
• GalNAc‐T2 attaches one sugar (GalNAc, a galactose derivative) to proteins
• It initiates protein glycosylation
N‐acetylgalactosaminyltransferase 2 (GalNAc‐T2)
GalNAc‐T2
2016
Carme Rovira
Structure of GalNAc‐T2
Catalyticdomain
Lectin domain
Linker
enzymeactive site
Collaboration withRamón Hurtado‐Guerrero(BIFI‐Zaragoza)
2016
Carme Rovira
Metadynamics simulation
The lectin domain “catches” the peptide and brings it to the catalytic site.
Lira‐Navarrete et al. Nat. Commun. 2015, 6, 6937.
Peptide binding to GalNAc‐T2 2016
Carme Rovira
Computer simulation
Atomic Force Microscopy
Lira‐Navarrete et al. Nat. Commun. 2015, 6, 6937.
Computationalmicroscope
Adapted from:DOI: 10.1021/acs.jcim.5b00249(A. Magistrato)
2016Carme Rovira
GalNAc‐T2 biochemical reaction
PEPTIDE
UDP
SUGAR
Angew. Chem. Int. Ed. 2014, 53, 8206
SUGAR TRANSFER
2016
Carme Rovira
Agraïments
Col∙laboradors teòrics:
Xevi Biarnés (IQS, Barcelona)
Víctor Rojas-Cervellera (Barcelona)
Albert Ardèvol (MPI, Frankfurt)
Javier Iglesias-Fernández (KCL, London)
Lluís Raich (UB, Barcelona)
Col∙laboradors experimentals:
Antoni Planas (IQS, Barcelona)
Peter Reilly (Iowa State U., USA)
Gideon J. Davies (U. York, UK)
Spencer Williams (Melbourne U., Australia)
Ramón Hurtado-Guerrero (BIFI, Zaragoza)
2016
Carme Rovira