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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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Protein dynamics and tunneling effects in the DHFR and TS catalysis

Amnon Kohen

Department of Chemistry

The University of Iowa


Overview
Overview catalysis

  • Background and experimental tools

  • Dihydrofolate Reductase (DHFR)

  • Dynamics-activity relationship

  • Thymidylate Synthase (TS)

  • Alternative TS (FDTS)


Uncatalyzed reaction

E catalysis

R.C.

A

C

+

+

B

D

Uncatalyzed reaction


Uncatalyzed catalysis vs. Enzyme-catalyzed reactions

E

R.C.


Kinetic complexity

E catalysis

R.C.

Kinetic complexity


Tunneling of a bound particle ground state nuclear tunneling

light isotope catalysis

Tunneling of a bound particleGround-State Nuclear Tunneling


Size catalysis

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.


A catalysisH/AT AD/AT

1.6 1.2

0.6 0.9

KIE Arrhenius Plots



Overview1
Overview catalysis

  • 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 catalysis

Radenine dinucleotide 2'-P

R'(p-aminobenzoyl)glutamate


Dhfr kinetics
DHFR Kinetics catalysis

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


O catalysis

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 catalysis

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 catalysis

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


O catalysis

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.


Determination of KIE catalysis

Fractional conversion determination:

Rt and R∞ determination:

for any time point (t) to (∞)

NADPH

NADP+

H4F

NADP+

NADPH


Coupled 1˚-2˚ motion catalysis

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?


Extracting intrinsic KIE from H/D/T catalysis

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

A catalysisl/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).


A catalysisH/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 Parameters catalysisInitial velocity of kcat at pH = 9


Overview2
Overview catalysis

  • 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 models catalysisMarcus-like model of ground-state tunneling



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.


MD calculations with DHFR motions in DHFR. The yellow arrows and arc indicate the coupled promoting motions.

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.


MD calculations with DHFR motions in DHFR. The yellow arrows and arc indicate the coupled promoting motions.

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.


MD calculations with DHFR motions in DHFR. The yellow arrows and arc indicate the coupled promoting motions.

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.


Dihydrofolate Reductase motions in DHFR. The yellow arrows and arc indicate the coupled promoting motions.

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


DHFR Temperature Dependency - w.t. vs. G121V motions in DHFR. The yellow arrows and arc indicate the coupled promoting motions.

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 models motions in DHFR. The yellow arrows and arc indicate the coupled promoting motions.Marcus-like model of ground-state tunneling


Overview3
Overview motions in DHFR. The yellow arrows and arc indicate the coupled promoting motions.

  • Background and experimental tools

  • Dihydrofolate Reductase (DHFR)

  • Dynamics-activity relationship

  • Thymidylate Synthase (TS)

  • Alternative TS (FDTS)


Dihydrofolate Reductase motions in DHFR. The yellow arrows and arc indicate the coupled promoting motions.


Thymidylate synthase
Thymidylate Synthase motions in DHFR. The yellow arrows and arc indicate the coupled promoting motions.


Synthesis of Labeled Substrates motions in DHFR. The yellow arrows and arc indicate the coupled promoting motions.


TS: Competitive Kinetic Assay motions in DHFR. The yellow arrows and arc indicate the coupled promoting motions.

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).


TS: motions in DHFR. The yellow arrows and arc indicate the coupled promoting motions. Arrhenius Plot of V/K KIEs: Observed

H/T

D/T


Arrhenius Plot of motions in DHFR. The yellow arrows and arc indicate the coupled promoting motions.V/K KIEs: Observed vs. Intrinsic

H/T

D/T


Arrhenius Plot of motions in DHFR. The yellow arrows and arc indicate the coupled promoting motions.V/K KIEs: Intrinsic

H/T

D/T


Arrhenius Plot of motions in DHFR. The yellow arrows and arc indicate the coupled promoting motions.V/K KIEs: Intrinsic

H/T

D/T


Semiclassically Calculated Range motions in DHFR. The yellow arrows and arc indicate the coupled promoting motions.

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.


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))


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))


A dataH/AT AD/AT

1.6 1.2

0.6 0.9

KIE Arrhenius Plots



Results and Conclusions data

  • 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 data

    • Background and experimental tools

    • Dihydrofolate Reductase (DHFR)

    • Dynamics-activity relationship

    • Thymidylate Synthase (TS)

    • Alternative TS (FDTS)


    Flavin dependent ts fdts
    Flavin Dependent TS (FDTS) data

    • 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?


    R data-[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 %)




    Flavin dependent ts
    Flavin dependent TS data

    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 data

    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 Truhlar Prof. Roald Hoffmann Prof. Chris Cheatum

    Jiali Gao

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



    Modeling data

    Reaction in D2O

    Flavin dependent TS

    Reaction with R-[6-T]MeTHF:

    All the T stay on the THF !

    (or at least 99.5%)


    -1 data

    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



    Jordi Villa` and Arieh Warshel* data

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


    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.


    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.


    Current real-time vibrationally adiabatic trajectory. (and 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.


    NMR relaxation studies real-time vibrationally adiabatic trajectory. (

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


    NMR relaxation studies real-time vibrationally adiabatic trajectory. (

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


    Theory: real-time vibrationally adiabatic trajectory. (

    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.


    Similar phenomenon was observed in non- enzymatic systems. real-time vibrationally adiabatic trajectory. (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 real-time vibrationally adiabatic trajectory. (

    Ki

    Ki = 280 ± 24 mM at 20 ˚C

    Ei = 22 ± 2 kcal/mol


    Example 1 real-time vibrationally adiabatic trajectory. (º (H/T) KIE Experiments


    Example 2 real-time vibrationally adiabatic trajectory. (º (H/T, D/T) KIE Experiments


    Kinetic Results real-time vibrationally adiabatic trajectory. (

    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.


    Extracting intrinsic KIE from H/D/T real-time vibrationally adiabatic trajectory. (

    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 real-time vibrationally adiabatic trajectory. (

    Intrinsic Isotope Effects in Enzymatic Reactions


    E real-time vibrationally adiabatic trajectory. (

    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.


    The Kuznetsov Ulstrup Formalism real-time vibrationally adiabatic trajectory. ((Mike Knapp and Judith Klinman)


    The kuznetsov ulstrup formalism mike knapp and judith klinman
    The Kuznetsov Ulstrup Formalism real-time vibrationally adiabatic trajectory. ((Mike Knapp and Judith Klinman)


    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.


    For recent reviews, see: tunneling. (Top) Environmental free energy surface, Q

    • 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.


    Summary tunneling. (Top) Environmental free energy surface, Q

    • 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


    Other Temperature-Independent KIEs tunneling. (Top) Environmental free energy surface, Q 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 tunneling. (Top) Environmental free energy surface, Q


    The temperature dependence of reaction rate1
    The Temperature Dependence of Reaction Rate tunneling. (Top) Environmental free energy surface, Q


    The temperature dependence of reaction rate2
    The Temperature Dependence of Reaction Rate tunneling. (Top) Environmental free energy surface, Q


    The temperature dependence of reaction rate3
    The Temperature Dependence of Reaction Rate tunneling. (Top) Environmental free energy surface, Q


    The temperature dependence of reaction rate4

    The Temperature Dependence of Reaction Rate tunneling. (Top) Environmental free energy surface, Q


    Semiclassically Calculated Range tunneling. (Top) Environmental free energy surface, Q

    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.


    Non-additive effects tunneling. (Top) Environmental free energy surface, Q

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


    Kinetic isotope effects in enzymatic reactions

    Commitments to Catalysis and Kinetic Complexity tunneling. (Top) Environmental free energy surface, Q

    Kinetic Isotope Effects in Enzymatic Reactions


    Kinetic Isotope Effects in Enzymatic Reactions tunneling. (Top) Environmental free energy surface, Q

    Commitments to Catalysis and Kinetic Complexity


    Kinetic complexity1

    3 tunneling. (Top) Environmental free energy surface, Q

    .

    2

    6

    k

    k

    H

    D

    <

    k

    k

    T

    T

    o

    b

    s

    o

    b

    s

    Kinetic Complexity


    ln( tunneling. (Top) Environmental free energy surface, Qk

    /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

    =


    What is the upper limit for the 2˚ exponent ( tunneling. (Top) Environmental free energy surface, Qy)

    with coupled motionbut notunneling(ysc)?

    Reduced mass considerationsysc = 4.25

    Vibrational analysis calculations ysc = 4.6

    Kinetic Complexity ysc = 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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