Amnon Kohen

1
Tunneling and Coupled Motion in Enzymatic
Catalysis
DHFR
Amnon Kohen Departments of Chemistry and Cell
and Molecular Biology The University of Iowa
2
Overview

• Background and experimental tools
• Dihydrofolate Reductase (DHFR)
• Distal mutations

3
Uncatalyzed reaction
4
Uncatalyzed vs. Enzyme-catalyzed reactions
E
R.C.
5
Kinetic Complexity
6
Tunneling of a bound particleGround-State
Nuclear Tunneling
7
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.

8
KIE Arrhenius Plots
9
Thymine Biosynthesis
Movie by Sawaya, M. R. and Kraut, J.
Biochemistry 1997, 36, 586-603.
10
(No Transcript)
11
Dihydrofolate Reductase
R?adenine dinucleotide 2'-P R'?(p-aminobenzoyl)glu
tamate
12
DHFR Kinetics
Fierke et al. Biochemistry (1987) 26, 4085-4092
13
Competitive KIE experiments with
DHFRMixed-labeled NADPH
H/T KIE
D/T KIE
14
Synthesis of Different Labeling Patterns for
theC4 Position of Nicotinamide Ring
15
Synthesis of Ad-14CC4-2H2 and Ad-14CC4-1H2
NADPH
16
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.

17
Determination of KIE
NADPH
2
1
NADP
H4F
NADP
NADPH
18
Example 2º (H/T, D/T) KIE Experiments
19
Coupled 1-2 motion? The mixed labeling
experiment
2 (H/T)H 1.19 0.015 2 (D/T)D 1.052
0.002

• 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?

20
Unsynchronized rehybridization
Pu,J., Ma,S., Garcia-Viloca, M., Gao,J.,
Truhlar,D.J., and Kohen, A. J. Am. Chem. Soc.
2005, 127, 14879-14886
21
Unsynchronized rehybridization
Pu,J., Ma,S., Garcia-Viloca, M., Gao,J.,
Truhlar,D.J., and Kohen, A. J. Am. Chem. Soc.
2005, 127, 14879-14886
22
Unsynchronized rehybridization
Pu,J., Ma,S., Garcia-Viloca, M., Gao,J.,
Truhlar,D.J., and Kohen, A. J. Am. Chem. Soc.
2005, 127, 14879-14886
23
(No Transcript)
24
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
25
(No Transcript)
26
(No Transcript)
27
(No Transcript)
28
(No Transcript)
29
(No Transcript)
30
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).
31
KIE Arrhenius Plots
32
DHFR Activation ParametersInitial velocity of
kcat at pH 9
33
Marcus Like Models Vibrationally Enhanced
Tunneling
34
Environmentally Coupled Tunneling
35
Tunneling Dynamics (PPV)Marcus-like model of
ground-state tunneling
36
Jordi Villa and Arieh Warshel J. Phys. Chem.B
2001, 7887-7907
37
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.
38
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.
39
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.
40
MD calculations with DHFR
Wong, K. F., Selzer, T., Benkovic, S. J., and
Hammes-Schiffer, S. Proc. Natl. Acad. Sci.
U.S.A. 2005 102, 6807-6812
41
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.
42
Dihydrofolate Reductase
Agarwal et al., PNAS 2002, 99, 2794-2799.
43
Effect of Remote Mutations
44
Effect of Remote Mutations
45
Effect of Remote Mutations
Effect of Remote Mutations
46
Effect of Remote Mutations
47
Effect of Remote Mutations
a
kH
S.C. Range
Al/Ah
48
Tunneling Dynamics (PPV)Marcus-like model of
ground-state tunneling
49
Conclusions and future directions

• 2 KIE provided no evidence for 1-2 coupled
  motion.
• Calculations reproducing 2 KIEs suggested that
  the rehybridization of the donor and acceptor is
  not synchronized.
• Intrinsic 1 KIEs calculated using the Northrop
  method seems to expose larger KIE than can be
  measured by pre-steady state methods.
• The temperature dependence and AL/AT of 1 KIEs,
  together with their size and activation
  parameters are in accordance with
  environmentally coupled tunneling models.
• Distal mutation presented non-additive
  (synergistic) effects which were in accordance
  with proposed dynamic network of motions across
  the protein - coupled to its catalyzed
  H-transfer.

50
Acknowledgments

• University of Iowa Scripps
• Dr. Kelli A. Markham Nitish Argawal Prof. Peter
  Kuhn
• Dr. R. Steve Sikorski Baoyu Hong Novartis
  (GNF)
• Lin Wang Dr. Cornelia Mihai Dr. Scott A.
  Lesley
• Scott Tharp Jigar Banderia UCSF
• 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 Irvin Dr. Frank Maley
• Prof. Stephen J. Benkovic Prof. Markus Ribbe
  Stanford
• Dr. Ravi Rajagopalan Dr. Yilin Hu Dr.
  Irimpan Matheos
• Dr. Tzvia Selzer Virginia Tech.
  University of Iowa
• U. Minnesota Prof. Dennis Dean Prof. Jan
  Jensen
• Donald Truhlar Texas AM Prof. Chris
  Cheatum
• Jiali Gao Prof. Michel Hall

NSF Career NIH-RO1 ACS-PRF NIH-R21 The
Frasch Foundation
51
http//cricket.chem.uiowa.edu/kohen/
52
(No Transcript)
53
Vibrationally Enhanced Tunneling
54
Vibrationally Enhanced Tunneling
Biophys. J.
1992
63, 689-699
Bruno W.J Bialek W.
55
The Kuznetsov Ulstrup Formalism(Mike Knapp and
Judith Klinman)
56
The Kuznetsov Ulstrup Formalism(Mike Knapp and
Judith Klinman)
57
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.
58
(No Transcript)
59
Dihydrofolate Reductase
Wong, K. F., Selzer, T., Benkovic, S. J., and
Hammes-Schiffer, S. Proc. Natl. Acad. Sci.
U.S.A. 2005 102, 6807-6812
60
Effect of Remote Mutations
61
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.
62
Relevant Protein Motion
63
DHFR Temperature Dependency - w.t. vs. G121V
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.
64
(No Transcript)
65
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.
66
NMR relaxation studies
Osborn et al., Biochemistry, 2001, 40, 9846-9859
67
NMR relaxation studies
Osborn et al., Biochemistry, 2001, 40, 9846-9859
68
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.
69
(No Transcript)
70
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.
71
(No Transcript)
72
Example 1º (H/T) KIE Experiments
73
Kinetic Results
Commitment 0.25
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.
74
Intrinsic Isotope Effects in Enzymatic Reactions
75
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.

76
(No Transcript)
77
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, 2458124587.
Temperature dependence for SBL. Klinman and
co-workers JACS (1996) 118, 10319-10320
78
(No Transcript)
79
(No Transcript)
80
The Temperature Dependence of Reaction Rate
81
The Temperature Dependence of Reaction Rate
82
The Temperature Dependence of Reaction Rate
83
The Temperature Dependence of Reaction Rate
84
The Temperature Dependence of Reaction Rate
85
(No Transcript)
86
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.
87
(No Transcript)
88
Non-additive effects
Rajagopalan et al., Biochemistry 2002, 41,
12618-12628
89
(No Transcript)
90
Kinetic Isotope Effects in Enzymatic Reactions
Commitments to Catalysis and Kinetic Complexity
91
Kinetic Isotope Effects in Enzymatic Reactions
Commitments to Catalysis and Kinetic Complexity
92
(No Transcript)
93
Kinetic Complexity
94
Extracting intrinsic KIE from H/D/T
95
(No Transcript)
96
Kinetic Isotope Effects (KIE) Semiclassical mass
dependence
Exp.

3.26