Transition state stabilization in enzyme catalysis: insights into mechanism from QM/MM modelling

AdrianJ. Mulholland

Centre for Computational Chemistry, School of Chemistry, University of Bristol, Bristol BS8 1TS, UK

GST: Mutations and stereospecificity

M2-2 isoenzyme produces only (S,S) product - why? Model effects of mutations (e.g. Ser209Ala, Asn8Asp and Thr13Ala represent differences between M1-1 and M2-2 GST isoenzymes)Calculate average changes in barrier and reaction energy through dynamicsAsn8Asp mutation identified as determinant of stereospecificity (increases tbarrier for forming the (9R,10R) product more than barrier for (9S,10S)Agrees with the fact that the M2-2 isoenzyme preferentially forms (9S,10S)Also identified role for Tyr115 in TS stabilization: important for catalysisSimulations can help understand and predict effects of polymorphism on GST specificity and stereospecificity

Introduction

References

Acknowledgements

'Insights into enzyme catalysis from QM/MM modelling: transition state stabilization in chorismate mutase' K.E. Ranaghan, L. Ridder, B. Szefczyk, W.A. Sokalski, J.C. Hermann & A.J. Mulholland, Mol. Phys., in press (2003). 'Identification of Glu166 as the General Base in the Acylation Reaction of Class A beta-Lactamases through QM/MM Modeling' Hermann, J. C.; Ridder, L.; Mulholland, A. J.; Holtje, H.-D.; J. Am. Chem. Soc.; in press (2003).

'Ab initio QM/MM modeling of the hydroxylation step in p-hydroxybenzoate hydroxylase' L. Ridder, J.N. Harvey, I. Rietjens, J. Vervoort & A.J. Mulholland, J. Phys. Chem. B, 107; 2118-2126 (2003). 'Modeling biotransformation reactions by combined quantum mechanical/molecular mechanical approaches: from structure to activity' L. Ridder & A.J. Mulholland, Current Topics in Medicinal Chemistry, 3, 1241-1256, (2003).

'QM/MM Free Energy Simulations of the Glutathione S-Transferase (M1-1) Reaction with Phenanthrene 9,10-Oxide' L. Ridder, I.Rietjens, J. Vervoort & A.J. Mulholland, J. Am. Chem. Soc., 124, 9926-9936 (2002). 'QM/MM Methods in Medicinal Chemistry', F. Perruccio, L. Ridder & A.J. Mulholland in 'Medicinal Quantum Chemistry', F. Alber & P. Carloni, Eds., Wiley, New York, (2003).

'The QM/MM Approach to Enzymatic Reactions' A.J. Mulholland, pp. 597-653 in 'Theoretical Biochemistry', L.Eriksson, Ed., Elsevier, Amsterdam, (2001). 'A QM/MM Study of the Hydroxylation of Phenol and Halogenated Derivatives by Phenol Hydroxylase' L. Ridder, A.J. Mulholland, I. Rietjens & J. Vervoort, J. Am. Chem. Soc.,122, 8728-8738 (2000).

'Ab Initio QM/MM Study of the Citrate Synthase Mechanism. A Low-Barrier Hydrogen Bond is Not Involved' A.J. Mulholland, P.D. Lyne & M. Karplus, J. Am. Chem. Soc., 122, 534-535 (2000).Role of Protein Flexibility in the Catalytic Cycle of p-Hydroxybenzoate Hydroxylase Elucidated by the Pro293Ser MutantB. A. Palfey, R. Basu, K.K. Frederick, K. B. Entsch, D.P. Ballou, Biochemistry, 41, 8438 (2002)

0

2

4

6

8

10

12

14

16

-3 -2 -1 0 1 2 3

Reaction coordinate (Angstrom)

QM

/MM

sta

bili

zati

on

en

erg

y (k

cal/m

ol,

AM

1/C

HA

RM

M)

TS-40.0

-30.0

-20.0

-10.0

0.0

10.0

20.0

30.0

40.0

-2.5 -1.5 -0.5 0.5 1.5

Reaction coordinate (A)

En

erg

y (k

cal/m

ol)

Reaction in solution(MP2/6-31+G(d)/PCM)Reaction in enzyme(MP2/6-31+G(d)//6-31G(d) QM/MM)

Substrate, catalytic residuestreated by QM

Glutathione S-Transferase (GST)Catalyses the addition of the tripeptide glutathione to endogenous and xenobiotic substratesPlays an important role in the detoxification and metabolism of xenobiotics (e.g. drugs)Exist as five classes (α, µ, π, κ, and θ) within which various isoenzymes are present Epoxide ring-opening of phenanthrene 9,10-oxide in M1-1 isoenzymeTwo possible alternative diastereomeric producs can be formed (R,R) and (S,S)An important model reaction for understanding determinants of stereospecificity in GSTsTwo possible alternative diastereomeric producs can be formed (R,R) and (S,S)An important model reaction for understanding determinants of stereospecificity in GSTs

Protein, solvent treated MM;interacts with QM

Free energy barrier is ~20 kcal/mol, compares well with experimental activation energy of ~18 Kcal/mol

M1-1 isoenzyme is not stereospecific: produces 50:50 mixture of (R,R) and (S,S) products (experiment again agrees with simulation findings)

Combined quantum mechanics/molecular mechanics (QM/MM) methodsallow modelling of reactions within enzymes

Substrate and glutathione modelled QM with reaction-specific AM1 parametersUmbrella sampling MD along approximate reaction coordinate (r(C-O)-r(S-C))

WHAM (Weighted Histogram Analysis Method) used to give Free Energy Profile

GST: QM/MM Dynamics Simulations

Model transition states and intermediates; identify and analyse key interactions

Calculations with ab initio, semiempirical or density functional QM

Molecular dynamics simulations for free energy profiles

Test and predict effects of mutations

Test mechanistic proposals

Small active site region treated QM (e.g 50 atoms)Surrounding protein and solvent (e.g. 25A radius)

treated by MM (e.g. CHARMM)

Recent applications demonstrate the useful insight QM/MM modelling can provide:

glutathione transferase (GST), chorismate mutase, and para-hydroxybenzoate hydroxylase (PHBH)

Tyr115

Ser209

Glutathionyl

Tyr6

Thr13

Phenan-threne9,10-oxide

Tyr115

Ser209

Glutathionyl

Tyr6

Thr13

Phenan-threne9,10-oxide

-20

-15

-10

-5

0

5

10

15

20

25

-3 -2 -1 0 1

Reaction coordinate (A)

Fre

e en

erg

y(k

cal/m

ol)

(9R,10R)

(9S,10S)

Successive simulations every 0.1A along reaction coordinate (e.g. 540ps total)

AJM thanks his co-workers and collaborators in the work described here (see references below). AJM is an EPSRC Advanced Research Fellow, and thanks EPSRC, BBSRC, the WellcomeTrust, the Wolfson Trust, the Royal Society and the European Union for support of this research.

Chorismate mutase

GST active site (TS complex)

Chorismate mutase active site (TS complex)

Claisen rearrangement of chorismate to prephenateNo covalent catalysis by the enzyme

Substrate strain/ the 'NAC' effect in catalysis?Ab initio and semiempirical QM/MM modellingComparison with reaction in solution

0

0.5

1

1.5

2

2.5

3

3.5

-1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4

reaction coordinate (A)

aver

age

nu

mbe

r o

f H

-bo

nd

s

(9S,10S)

(9R,10R)

average

GST: solvation of thiolate sulfur along reaction path

Para-hydroxybenzoate hydroxylase (PHBH)Biodegradation of aromatic compounds (pollutants etc)Flavoprotein monoxygenase; hydroperoxyflavin intermediateAb initio and semiempirical QM/MMElectrophilic aromatic substitution mechanism

Wide range of halogenated substrates

-10

-8

-6

-4

-2

0

2

4

-2 -1.5 -1 -0.5 0 0.5 1 1.5

reaction coordinate (A)en

erg

y (k

cal/m

ol)

Effect of whole proteinEffect of Pro293 carbonyl

-80

-60

-40

-20

0

20

40

-2 -1.5 -1 -0.5 0 0.5 1

reaction coordinate (A)

QM

en

erg

y (k

cal/m

ol)

HF/3-21G*

B3LYP/6-311+G** // HF/3-21G*

LMP2/6-31+G* // HF/3-21G*

AM1

PHBH active site showing transition stateinteraction with Pro293

PHBHCalculations identified novel catalytic interactionHydrogen bond with Pro293 carbonylSpecifically stabilizes transition state (only)Supported by subsequent experiments

Now also found in phenol hydroxylase (PH)General feature of flavoprotein oxygenase catalysis?

QM/MM tested by higher level calculations

PHBH

PHBH

Calculated GST free energy profiles

Correlation between experimental and calculated barriers

Validates QM/MM modelIdentification of mechanismRate-limiting step

Correlation with experimental rates for phenol hydroxylase also

4

5

6

7

8

9

17 18 19 20 21 22 23

COO

O

F

COO

O F

COO

O COO

O F F

COO

O

F

F

F

F

ln k

cat

QM/MM energy barrier (kcal/mol)

R = 0.91

3

4

5

6

7

23 24 25 26 27 28 29 30 31

QM/MM energy barrier (kcal/mol)

ln k

cat

Phenol hydroxylase (PH):Predict rates for alternative substrates

PH

Stabilization by the enzyme is greatest at the transition stateEnzyme binds TS more strongly than substrateTS stabilization central to catalysis

TS is stabilized (relative to substrate) more by the enzyme than it is in solution

Calculated rate acceleration consistent with experiment

Substrate is distorted: strain/compression may also assist catalysis

Chorismate mutase active site is complementary to the transition state

Major contributions to TS stabilization from Arg90, Glu78,Arg7, and some bound water molecules

ConclusionsQM/MM modelling can provide detailed insight into enzyme mechanismsCan compare mechanisms, model transition statesIdentify and analyse catalytic interactionsResults demonstrate the importance of TS stabilizationNovel TS stabilizing interaction identified in PHBH and PHRelative stabilities of TSs determine stereospecificity in GSTChorismate mutase active site is exquisitely well-organized to stabilize TSModelling helps relate enzyme structure to activity

OH OH

OHO2

COOH COOH

OH OH

OHO2

PHBH:

Chorismate mutase: stabilization along reaction path