Protein dynamics and tunneling effects in the DHFR and TS catalysis
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Protein dynamics and tunneling effects in the DHFR and TS catalysis. Amnon Kohen Department of Chemistry The University of Iowa. Overview. Background and experimental tools Dihydrofolate Reductase (DHFR) Dynamics-activity relationship Thymidylate Synthase (TS) Alternative TS (FDTS). E.

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Amnon kohen department of chemistry the university of iowa

Protein dynamics and tunneling effects in the DHFR and TS catalysis

Amnon Kohen

Department of Chemistry

The University of Iowa


Overview

Overview

  • Background and experimental tools

  • Dihydrofolate Reductase (DHFR)

  • Dynamics-activity relationship

  • Thymidylate Synthase (TS)

  • Alternative TS (FDTS)


Uncatalyzed reaction

E

R.C.

A

C

+

+

B

D

Uncatalyzed reaction


Amnon kohen department of chemistry the university of iowa

Uncatalyzed vs. Enzyme-catalyzed reactions

E

R.C.


Kinetic complexity

E

R.C.

Kinetic complexity


Tunneling of a bound particle ground state nuclear tunneling

light isotope

Tunneling of a bound particleGround-State Nuclear Tunneling


Amnon kohen department of chemistry the university of iowa

Size

H

[product]

D

time

Temperature dependency

AH/AT AD/AT

1.6 1.2

0.6 0.9

KIEs as Probe of Tunneling

  • Swain, C. G. et al., J. Am. Chem. Soc.1958, 80, 5885-5893

  • Huskey, W. P.; Schowen, R. L. J. Am. Chem. Soc.1983, 105, 5704-5706.

  • Saunders, W. H. J. Am. Chem. Soc.1985, 107, 164-169.

  • Kohen, A.* and Jensen J.H. J. Am. Chem. Soc. 2002,124, 3858-3864.

  • Kohen, A.*Prog. React. Kin. Mech.2003, 28, 119-156.


Amnon kohen department of chemistry the university of iowa

AH/AT AD/AT

1.6 1.2

0.6 0.9

KIE Arrhenius Plots


Amnon kohen department of chemistry the university of iowa

Thymine biosynthesis


Overview1

Overview

  • Background and experimental tools

  • Dihydrofolate Reductase (DHFR)

  • Dynamics-activity relationship

  • Thymidylate Synthase (TS)

  • Alternative TS (FDTS)

Movie by Sawaya, M. R. and Kraut, J. Biochemistry 1997, 36, 586-603.


Dihydrofolate reductase

Dihydrofolate Reductase

Radenine dinucleotide 2'-P

R'(p-aminobenzoyl)glutamate


Dhfr kinetics

DHFR Kinetics

Fierke et al. Biochemistry (1987) 26, 4085-4092


Amnon kohen department of chemistry the university of iowa

O

O

O

O

H

T

D

T

T

D

T

H

O

O

D

D

H

H

N

H

N

H

N

H

N

H

2

2

2

2

N

H

N

H

2

2

N

N

N

N

N

N

=

=

=

R

O

P

O

=

R

O

P

O

R

O

P

O

R

O

P

O

3

3

3

3

=

=

R

O

P

O

R

O

P

O

*

*

4

S

-

[

H

,

T

]

-

N

A

D

P

H

3

3

4

S

-

[

D

,

T

]

-

N

A

D

P

H

4

R

-

[

D

,

T

]

-

N

A

D

P

H

4

R

-

[

H

,

T

]

-

N

A

D

P

H

1

4

1

4

[

A

d

-

C

]

N

A

D

P

H

[

A

d

-

C

]

N

A

D

P

H

Competitive KIE experiments with DHFRMixed-labeled NADPH

H/T KIE

D/T KIE


Synthesis of different labeling patterns for the c 4 position of nicotinamide ring

GDH

Glucose-1-D

GDH

Glucose-1-T

GDH

Glucose-1-H

O

O

O

O

H

T

D

T

T

D

T

H

N

H

N

H

N

H

N

H

2

2

2

2

N

N

N

N

=

=

=

R

O

P

O

=

R

O

P

O

R

O

P

O

R

O

P

O

3

3

3

3

4

S

-

[

H

,

T

]

-

N

A

D

P

H

4

S

-

[

D

,

T

]

-

N

A

D

P

H

4

R

-

[

D

,

T

]

-

N

A

D

P

H

4

R

-

[

H

,

T

]

-

N

A

D

P

H

Synthesis of Different Labeling Patterns for theC4 Position of Nicotinamide Ring


Synthesis of ad 14 c c 4 2 h 2 and ad 14 c c 4 1 h 2 nadph

GDH

glucose-1-D

GDH

glucose-1-H

O

O

D

D

H

H

N

H

N

H

2

2

N

N

=

=

R

O

P

O

R

O

P

O

*

*

3

3

1

4

1

4

[

A

d

-

C

]

N

A

D

P

H

[

A

d

-

C

]

N

A

D

P

H

Synthesis of [Ad-14C;C4-2H2] and [Ad-14C;C4-1H2] NADPH


Amnon kohen department of chemistry the university of iowa

O

O

O

O

H

T

D

T

T

D

T

H

O

O

D

D

H

H

N

H

N

H

N

H

N

H

2

2

2

2

N

H

N

H

2

2

N

N

N

N

N

N

=

=

=

R

O

P

O

=

R

O

P

O

R

O

P

O

R

O

P

O

3

3

3

3

=

=

R

O

P

O

R

O

P

O

*

*

4

S

-

[

H

,

T

]

-

N

A

D

P

H

3

3

4

S

-

[

D

,

T

]

-

N

A

D

P

H

4

R

-

[

D

,

T

]

-

N

A

D

P

H

4

R

-

[

H

,

T

]

-

N

A

D

P

H

1

4

1

4

[

A

d

-

C

]

N

A

D

P

H

[

A

d

-

C

]

N

A

D

P

H

Competitive KIE experiments with DHFRMixed-labeled NADPH

H/T KIE

D/T KIE

  • Markham et al., (2003) Anal. Biochem.322, 26-32.

  • Agrawal, N., and Kohen, A. (2003) Anal. Biochem.322, 179-184

  • Markham et al., (2004) Anal. Biochem., 325, 62-67.

  • McCracken et al., (2003) Anal. Biochem., 324, 131-136.


Amnon kohen department of chemistry the university of iowa

Determination of KIE

Fractional conversion determination:

Rt and R∞ determination:

for any time point (t) to (∞)

NADPH

NADP+

H4F

NADP+

NADPH


Amnon kohen department of chemistry the university of iowa

Coupled 1˚-2˚ motion

From the mixed labeling experiment

Ln(1.19)/ln(1.052)=3.4 ±1 —No coupled motion

Calculated vs. experimental 2˚ H/D KIEs

Calculated : 1.13 Mireia Garcia-Viloca, Donald G. Truhlar,* and Jiali Gao* Biochemistry 2003, 42, 13558-13575

Experimental: 1.13 ± 0.02

Equilibrium: 1.127 ± 0.009

Location of the transition state?


Amnon kohen department of chemistry the university of iowa

Extracting intrinsic KIE from H/D/T

H/D/T data allow calculations of an intrinsic KIE:

Northrop, D.B. In Enzyme mechanism from isotope effects; Cook, P. F., Ed.; CRC Press: Boca Raton, Fl., 1991, pp 181-202.

http://cricket.chem.uiowa.edu/~kohen/tools.html


Temperature dependence as a criterion for tunneling

Al/Ah

Upper Limits Al/Ah*

H/D

3.50.5

1.4

H/T

7.01.5

1.6

D/T

1.700.14

1.2

Temperature Dependence as a Criterion for Tunneling

Schneider & Stern (1972) J.A.C.S., 94, 1517-1522.

Stern & Weston, (1974) J.Chem. Phys.., 60, 2815-2821.

Bell (1980) The Tunneling Effect in Chemistry, Chapman & Hall, ED., London & New York.

Melander & Saunders (1987) Reactions Rates of Isotopic Molecules, Krieger, Ed., Fl.

Sikorski, R. S., Wang, L., Markham, K. A., Rajagopalan, P. T. R., Benkovic, S. J., and Kohen, A.*

J. Am. Chem. Soc., 126, 4778-4779 (2004).


Amnon kohen department of chemistry the university of iowa

AH/AT AD/AT

1.6 1.2

0.6 0.9

KIE Arrhenius Plots


Dhfr activation parameters initial velocity of k cat at ph 9

DHFR: Activation ParametersInitial velocity of kcat at pH = 9


Overview2

Overview

  • Background and experimental tools

  • Dihydrofolate Reductase (DHFR)

  • Dynamics-activity relationship

  • Thymidylate Synthase (TS)

  • Alternative TS (FDTS)


Tunneling dynamics in theoretical models marcus like model of ground state tunneling

Tunneling & Dynamics in theoretical modelsMarcus-like model of ground-state tunneling


Amnon kohen department of chemistry the university of iowa

Vibrationally Enhanced Tunneling


Amnon kohen department of chemistry the university of iowa

Diagram of a portion of the network of coupled promoting motions in DHFR. The yellow arrows and arc indicate the coupled promoting motions.

Benkovic, Hammes-Shiffer and co-workers PNAS (2002) 99, 2794-2799.


Amnon kohen department of chemistry the university of iowa

MD calculations with DHFR

Jennifer L. Radkiewicz and Charles L. Brooks, III* J. Am. Chem. Soc. 2000, 122, 225-231.

(a) DHFR/DHF/NADPH

Figure 5. Residue-residue based map of correlated motions. Red and yellow indicate regions of positive correlation, and dark blue indicates regions of anti-correlation.


Amnon kohen department of chemistry the university of iowa

MD calculations with DHFR

Jennifer L. Radkiewicz and Charles L. Brooks, III* J. Am. Chem. Soc. 2000, 122, 225-231.

(a) DHFR/DHF/NADPH

(b) DHFR/THF/NADP+

Figure 5. Residue-residue based map of correlated motions. Red and yellow indicate regions of positive correlation, and dark blue indicates regions of anti-correlation.


Amnon kohen department of chemistry the university of iowa

MD calculations with DHFR

Jennifer L. Radkiewicz and Charles L. Brooks, III* J. Am. Chem. Soc. 2000, 122, 225-231.

(a) DHFR/DHF/NADPH

(b) DHFR/THF/NADP+

(c) DHFR/THF/NADPH

Figure 5. Residue-residue based map of correlated motions. Red and yellow indicate regions of positive correlation, and dark blue indicates regions of anti-correlation.


Amnon kohen department of chemistry the university of iowa

Dihydrofolate Reductase

Agarwal et al., PNAS 2002, 99, 2794-2799.


Amnon kohen department of chemistry the university of iowa

DHFR Temperature Dependency - w.t. vs. G121V

Commitment

3.2±0.3

H

3.7±0.2

Ea in kcal/mol

At high and low temperature

7.3±0.5

H

11.9±0.5

D

D

2.5±1.2

3.1±0.4

9.2±2.1

7.5±0.7

G121V:

Wild Type:

Intrinsic KIEs

Observed KIEs

Observed H/D on kcat

Observed H/D on kcat

Pre-steady-state KIE

Intrinsic KIEs were calculated following:Northrop, D. B. In Enzyme mechanism from isotope effects; Cook, P. F., Ed.; CRC Press, 1991, pp 181-202.


Tunneling dynamics in theoretical models marcus like model of ground state tunneling1

Tunneling & Dynamics in theoretical modelsMarcus-like model of ground-state tunneling


Overview3

Overview

  • Background and experimental tools

  • Dihydrofolate Reductase (DHFR)

  • Dynamics-activity relationship

  • Thymidylate Synthase (TS)

  • Alternative TS (FDTS)


Amnon kohen department of chemistry the university of iowa

Dihydrofolate Reductase


Thymidylate synthase

Thymidylate Synthase


Amnon kohen department of chemistry the university of iowa

Synthesis of Labeled Substrates


Amnon kohen department of chemistry the university of iowa

TS: Competitive Kinetic Assay

H/T and D/T KIEs were measured. Intrinsic KIEs were calculated and their temperature dependence determined at 5-45 ˚C range.

Nitish Agrawal, Cornelia Mihai, and Amnon Kohen*, Anal. Biochem.328, 44-50 (2004).

Agrawal, N., Hong, B., Mihai, C., and Kohen, A.*Biochemistry, 43, 1998-2006 (2004).


Amnon kohen department of chemistry the university of iowa

TS: Arrhenius Plot of V/K KIEs: Observed

H/T

D/T


Amnon kohen department of chemistry the university of iowa

Arrhenius Plot of V/K KIEs: Observed vs. Intrinsic

H/T

D/T


Amnon kohen department of chemistry the university of iowa

Arrhenius Plot of V/K KIEs: Intrinsic

H/T

D/T


Amnon kohen department of chemistry the university of iowa

Arrhenius Plot of V/K KIEs: Intrinsic

H/T

D/T


Amnon kohen department of chemistry the university of iowa

Semiclassically Calculated Range

for the KIE on Arrhenius Preexponential Factors AH/AT and AD/AT

Schneider & Stern (1972) J.A.C.S., 94, 1517-1522.

Stern & Weston, (1974) J.Chem. Phys.., 60, 2815-2821.

Bell (1980) The Tunneling Effect in Chemistry, Chapman & Hall, ED., London & New York.

Melander & Saunders (1987) Reactions Rates of Isotopic Molecules, Krieger, Ed., Fl.


Amnon kohen department of chemistry the university of iowa

TS : Temperature dependence of steady-state initial velocity data

40°C

30°C

20°C

5°C

Values of the kcat were determined by fitting steady-state

initial velocity data to the following substrate inhibition equation:

V = kcat[S]/(Km + [S]* (1+[S]2/KS))


Amnon kohen department of chemistry the university of iowa

TS : Temperature dependence of steady-state initial velocity data

40°C

30°C

20°C

5°C

1.8

Ea = 4 ± 0.1 Kcal/mol

40°C

30°C

20°C

5°C

Values of the kcat were determined by fitting steady-state

initial velocity data to the following substrate inhibition equation:

V = kcat[S]/(Km + [S]* (1+[S]2/KS))


Amnon kohen department of chemistry the university of iowa

AH/AT AD/AT

1.6 1.2

0.6 0.9

KIE Arrhenius Plots


Relevant protein motion

Relevant Protein Motion


Amnon kohen department of chemistry the university of iowa

Results and Conclusions

  • DHFR:

    • 1.The lack of temperature dependence of intrinsic primary KIEs constitutes proof for hydrogen tunneling, and taken together with the Ea suggests vibrationally enhanced H-tunneling.

    • 2. The secondary intrinsic KIEs were 1.19 ±0.015 and 1.052 ±0.019 for H/T and D/T, respectively, which doesn’t suggest 1˚-2˚ coupled motion (the Swain-Schaad EXP = 3.4 ±1).

    • 3. The intrinsic KIEs at 25 ˚C are in agreement with the values predicted by Truhlar, Gao, Hammes-Schiffer and their co-workers from QM/MM calculations.

    • 4. The dynamically altered mutant G121V catalyze similar H-transfer mechanism but its pre-organization is not perfect and some “gating” is required.

  • * Sikorski, et al., J. Am. Chem. Soc., 126, 4778-4779 (2004).

  • TS:

    • 1. The lack of temperature dependence of intrinsic primary KIEs constitutes proof for hydrogen tunneling, and taken together with the Ea suggests vibrationally enhanced H-tunneling.

    • 2. Substrate inhibition was predicted but is observed here for the first time. This demonstrates that relevant activation parameters can only be calculated on a single rate constant (e.g., kcat) if it is extracted from the whole kinetic cascade at all temperatures.

    • * Agrawal, et al., . Biochemistry, 43, 1998-2006 (2004).


  • Overview4

    Overview

    • Background and experimental tools

    • Dihydrofolate Reductase (DHFR)

    • Dynamics-activity relationship

    • Thymidylate Synthase (TS)

    • Alternative TS (FDTS)


    Flavin dependent ts fdts

    Flavin Dependent TS (FDTS)

    • The ThyA gene that encodes for thymidylate synthase (TS) is absent in the genomes of a large number of bacteria, including several human pathogens.

    • Many of these bacteria also lack the genes for dihydrofolate reductase (DHFR) and thymidine kinase, and are totally dependent on an alternative enzyme for thymidylate synthesis.

    • Thy1 encodes flavin-dependent TS (FDTS) and shares no sequence homology or structure similarities with classical TS.

    • Same reactants as TS but dTMP and THF products

    • We studied the mechanism of a FDTS from Thermotoga maritima (TM0449).

    Is it a bifunctional TS-DHFR or an entirely different mechanism?


    Amnon kohen department of chemistry the university of iowa

    R-[4-3H]-NADPH and [2-14C]-dUMP

    S-[4-3H]-NADPH and [2-14C]-dUMP

    Flavin dependent TS

    Reaction with R-[6-T]MeTHF:

    All the T stay on the THF !

    (or at least 99.7 %)


    Amnon kohen department of chemistry the university of iowa

    Flavin dependent TS


    Amnon kohen department of chemistry the university of iowa

    Flavin dependent TS


    Flavin dependent ts

    Flavin dependent TS

    Summary

    • The reaction proceeds via a Ping Pong mechanism where nicotinamide binding and release precedes the oxidative half reaction.

    • The enzyme is primarily pro-R specific with regard to the nicotinamide (NADPH), whose oxidation is the rate limiting step of the whole catalytic cascade.

    • An enzyme bound flavin is reduced with an isotope effect of ~25 (consistent with H-tunneling), and exchanges protons with the solvent prior to the reduction of an intermediate methylene.

    • A significant NADPH substrate-inhibition and large KM rationalized the slow activity reported for this enzyme in the past. Is HADPH the natural reductant?

    • A new mechanism was proposed that is very different from that of classical TSs.

    • The differences between the FDTS proposed mechanism and that of the classical TS invoke the notion that mechanism based drugs will selectively inhibit FDTS and will not have much effect on human (and other eukaryotes) TS.

    Agrawal, N., Lesley, S., Kuhn, and Kohen, A.*Biochemistry,43, 10295-10301 (2004).


    Acknowledgments

    Acknowledgments

    University of IowaScripps

    Kelli A. Markham Nitish ArgawalProf. Peter Kuhn

    Dr. R. Steve Sikorski Baoyu HongNovartis (GNF)

    Lin Wang Dr. Cornelia Mihai Dr. Scott A. Lesley

    Scott Tharp Jigar BanderiaUCSF

    Malia Moore Dr.Amandeep K. Sra Prof. Robert Stroud

    Jocelyn McCracken Dr.Anatoly Chernyshev Dr. Pat Green

    Todd Fleischmann NY State Dept. Health

    Penn. State U.UC IrvinDr. Frank Maley

    Prof. Stephen J. BenkovicProf. Markus Ribbe Stanford

    Dr. Ravi RajagopalanVirginia Tech.Dr. Irimpan Matheos

    Dr. Tzvia SelzerProf. Dennis Dean University of Iowa

    U. MinnesotaCornell UniversityProf. JanJensen

    Donald TruhlarProf. Roald Hoffmann Prof. Chris Cheatum

    Jiali Gao

    NSF Career; NIH-RO1; ACS-PRF; NIH-R21; The Frasch Foundation


    Amnon kohen department of chemistry the university of iowa

    http://cricket.chem.uiowa.edu/~kohen/


    Amnon kohen department of chemistry the university of iowa

    Modeling

    Reaction in D2O

    Flavin dependent TS

    Reaction with R-[6-T]MeTHF:

    All the T stay on the THF !

    (or at least 99.5%)


    Amnon kohen department of chemistry the university of iowa

    -1

    kcat= 0.1 s

    KM= 4 mM

    Flavin dependent TS

    Substrate inhibition at 37 ˚C:

    v = kcat[S]/(KM + [S](1+[S]/KS))

    Dithionite has kcatof 7 s

    -1

    KMs for MeTHF and dUMP with dithionite are ~0.04 mM


    Amnon kohen department of chemistry the university of iowa

    Vibrationally Enhanced Tunneling


    Amnon kohen department of chemistry the university of iowa

    Jordi Villa` and Arieh Warshel*

    J. Phys. Chem.B 2001, 7887-7907


    Amnon kohen department of chemistry the university of iowa

    Vibrational wave functions of the transferring hydride for representative configurations. On the donor side, the donor carbon atom and its first neighbors are shown, whereas on the acceptor side, the acceptor carbon atom and its first neighbors are shown. The ground and excited vibrational states are shown on the left and right, respectively.

    Hammas-Shiffer and co-workers

    J. Phys.Chem. B (2002) 106, 8283-8293.


    Amnon kohen department of chemistry the university of iowa

    Time evolution of two select distances for a representative real-time vibrationally adiabatic trajectory. (A) Donor-acceptor distance. (B) Distance between Ca of Gly 121 and Cb of Met-42.

    Equilibrium averages of geometrical properties along the collective reaction coordinate.

    Benkovic, Hammes-Shiffer and co-workers PNAS (2002) 99, 2794-2799.


    Amnon kohen department of chemistry the university of iowa

    Currentand Future Directions

    • Methods were developed that expose the nature of the chemical steps in the DHFR and TS reactions. Tools are being developed to study different C-H bond activation with these enzymes.

    • Various DHFR mutants with altered dynamics are studied to examine possible effects of those dynamics on the nature of the hydride transfer (e.g., G121V, M42W, and G121V-M42W).

    • Alternative DHFRs and TSs, such as R67-DHFR, thermophiles and halophiles, will be studied and the nature of their chemical transformations will be compared. The relationship between sequence, structure, dynamics and function (H-transfer catalysis) will be examined.

    • FDTS from T. maritima and its mutants are being studied to further explore its mechanism. All experiments are also preformed at 65 ˚C.


    Amnon kohen department of chemistry the university of iowa

    NMR relaxation studies

    Osborn et al., Biochemistry, 2001, 40, 9846-9859


    Amnon kohen department of chemistry the university of iowa

    NMR relaxation studies

    Osborn et al., Biochemistry, 2001, 40, 9846-9859


    Amnon kohen department of chemistry the university of iowa

    Theory:

    Network of coupled promoting motions in enzyme catalysis

    A network of coupled promoting motions in the enzyme DHFR is identified based on genomic analysis for sequence conservation, kinetic measurements of multiple mutations, and mixed quantum-classical molecular dynamics simulations of hydride transfer.

    The motions in this network span time scales of fs to ms and are found on the exterior of the enzyme as well as in the active site.

    Benkovic, Hammes-Shiffer and co-workers PNAS (2002) 99, 2794-2799.


    Amnon kohen department of chemistry the university of iowa

    Similar phenomenon was observed in non- enzymatic systems.Yet, a great way to look into the nature of the chemical step in complex kinetic cascades (e.g., enzymatic systems).Kohen, ,A Prog. React. Kin. Mech. (2003) 28, 119-156.


    Ts bibi ordered binding and release

    TS: BiBi ordered binding and release

    Ki

    Ki = 280 ± 24 mM at 20 ˚C

    Ei = 22 ± 2 kcal/mol


    Amnon kohen department of chemistry the university of iowa

    Example 1º (H/T) KIE Experiments


    Amnon kohen department of chemistry the university of iowa

    Example 2º (H/T, D/T) KIE Experiments


    Amnon kohen department of chemistry the university of iowa

    Kinetic Results

    Commitment: 0.25

    Ln(1.19)/ln(1.052)=3.3 ±0.5 —No coupled motion

    a. Calculated using the methodology developed by Dexter B. Northrop (Ref: Northrop, D. B. In Enzyme mechanism from isotope effects; Cook, P. F., Ed.; CRC Press: Boca Raton, Fl., 1991, pp 181-202).

    b. Calculated using the commitment for protium taking into account protium contamination in D/T experiments.


    Amnon kohen department of chemistry the university of iowa

    Extracting intrinsic KIE from H/D/T

    E

    R.C.

    3

    3

    .

    .

    2

    2

    6

    6

    k

    k

    k

    k

    H

    H

    D

    D

    <

    k

    =

    k

    k

    k

    T

    T

    T

    T

    o

    b

    s

    o

    b

    s


    Intrinsic isotope effects in enzymatic reactions

    http://cricket.chem.uiowa.edu/~kohen/tools.html

    Intrinsic Isotope Effects in Enzymatic Reactions


    Amnon kohen department of chemistry the university of iowa

    E

    l

    s

    l

    eq

    x

    Where

    l

    l

    s

    is the barrier width at which the hydrogen may tunnels and

    is the equilibrium width.

    eq

    2

    2

    S'

    -1/2

    S'

    1/2

    2

    3

    ln(k

    / k

    )

    = -

    ln(m) +

    1- (m)

    2S -

    + (m)

    -1

    L

    T

    4

    S"

    S"

    

    T

    1/k

    L

    H or D; m

    1or 2; S

    the WKB action;

    B

    Vibrationally Enhanced Tunneling

    Biophys. J.

    1992

    63, 689-699

    Bruno W.J & Bialek W.


    Amnon kohen department of chemistry the university of iowa

    The Kuznetsov Ulstrup Formalism(Mike Knapp and Judith Klinman)


    The kuznetsov ulstrup formalism mike knapp and judith klinman

    The Kuznetsov Ulstrup Formalism(Mike Knapp and Judith Klinman)


    Amnon kohen department of chemistry the university of iowa

    Energy surface for environmentally coupled hydrogen tunneling. (Top) Environmental free energy surface, Qenv, with the free energy of reaction (DG˚) and reorganization energy (k) indicated. (Bottom) hydrogen potential energy surface, qH, at different environmental configurations. R0 is the reactant configuration, ‡ denotes the reactive configuration, and P0 is the product configuration. Gating also alters the distance (Dr) of hydrogen transfer.


    Amnon kohen department of chemistry the university of iowa

    For recent reviews, see:

    • Schowen, Eur. J. Biochem., (2002) 269, 3095.

    • Sutcliffe and Scrutton, Eur. J. Biochem., (2002) 269, 3096.

    • Antoniou et al., Eur. J. Biochem., (2002) 269, 3103.

    • Knapp and Klinman, Eur. J. Biochem., (2002) 269, 3113.

    • Comment:

    • The tunneling promoting effect of environmental dynamics was suggested from kinetic measurements.

    • Experimental probes for vibrational dynamics with proteins are quite challenging.


    Amnon kohen department of chemistry the university of iowa

    Summary

    • Tunneling’s role in enzyme catalysis:

      • Probably significant, yet, model dependent.

      • Does it contribute to Catalysis?

    • More studies of relevant non-enzymatic systems are required.

    • The role of enzyme’s dynamics in catalysis:

      • Nomenclature.

      • Experimental work that correlate dynamics to kinetics. (Which dynamics? Coupling mechanisms).

    • Comparison to solution and model reactions


    Amnon kohen department of chemistry the university of iowa

    Other Temperature-Independent KIEs in Enzyme Catalysis

    H

    H

    D

    D

    Temperature dependence and KIE data for H172Q TMADH.

    Basran, Sutcliffe and Scrutton

    JBC (2001) 276, 24581–24587.

    Temperature dependence for SBL.

    Klinman and co-workers

    JACS (1996) 118, 10319-10320


    The temperature dependence of reaction rate

    The Temperature Dependence of Reaction Rate


    The temperature dependence of reaction rate1

    The Temperature Dependence of Reaction Rate


    The temperature dependence of reaction rate2

    The Temperature Dependence of Reaction Rate


    The temperature dependence of reaction rate3

    The Temperature Dependence of Reaction Rate


    The temperature dependence of reaction rate4

    The Temperature Dependence of Reaction Rate


    Amnon kohen department of chemistry the university of iowa

    Semiclassically Calculated Range

    for the KIE on Arrhenius Preexponential Factors AH/AT and AD/AT

    • AH/AT AD/AT

  • Upper limit 1.6 1.2

  • Lower limit 0.6 0.9

  • Schneider & Stern (1972) J.A.C.S., 94, 1517-1522.

    Stern & Weston, (1974) J.Chem. Phys.., 60, 2815-2821.

    Bell (1980) The Tunneling Effect in Chemistry, Chapman & Hall, ED., London & New York.

    Melander & Saunders (1987) Reactions Rates of Isotopic Molecules, Krieger, Ed., Fl.


    Amnon kohen department of chemistry the university of iowa

    Non-additive effects

    Rajagopalan et al., Biochemistry 2002, 41, 12618-12628


    Kinetic isotope effects in enzymatic reactions

    Commitments to Catalysis and Kinetic Complexity

    Kinetic Isotope Effects in Enzymatic Reactions


    Amnon kohen department of chemistry the university of iowa

    Kinetic Isotope Effects in Enzymatic Reactions

    Commitments to Catalysis and Kinetic Complexity


    Kinetic complexity1

    3

    .

    2

    6

    k

    k

    H

    D

    <

    k

    k

    T

    T

    o

    b

    s

    o

    b

    s

    Kinetic Complexity


    Amnon kohen department of chemistry the university of iowa

    ln(k

    /k

    )

    1/2

    1/2

    1/(m

    )

    - 1/(m

    )

    H

    T

    D

    T

    1/2

    1/2

    ln(k

    / k

    )

    1/(m

    )

    - 1/(m

    )

    D

    T

    H

    T

    Kinetic Isotope Effects (KIE)

    Semiclassical mass dependence

    Exp.

    =

    =

    3.26

    =


    Amnon kohen department of chemistry the university of iowa

    What is the upper limit for the 2˚ exponent (y)

    with coupled motionbut notunneling(ysc)?

    Reduced mass considerationsysc = 4.25

    Vibrational analysis calculationsysc = 4.6

    Kinetic Complexityysc = 4.8

    Experimental EXP larger4.8indicates tunneling

    Yet, any EXP > 3.3 could suggest tunneling

    Several measurements may support tunneling

    at the 3.3<EXP<4.8 range

    Kohen, A.* and Jensen J.J. Am. Chem. Soc. (2002), 124, 3858.


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