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1
By
Arren Bar-Even
Advisor:
Ron Milo
21 March 2012
Design Principles of Cellular Metabolism
-
:
Thesis for the degree
Doctor of Philosophy
Submitted to the Scientific Council of the Weizmann Institute of Science
Rehovot, Israel
( )
,
2
Table of contents
Page
Acknowledgments 3
Abbreviations 4
Summary of Findings 5
Summary of Findings (Hebrew) 6
Introduction 7
Methods 16
Results
1. Evolutionary and physicochemical trends shaping enzyme parameters 24
2. Hydrophobicity and charge shape cellular metabolite concentrations 31
3. Quantitative pathway analysis applied to carbon fixation 36
4. Thermodynamic constraints shape the structure of carbon fixation pathways 42
5. The design of synthetic carbon fixation pathways 55
6. Rethinking glycolysis: a perspective on the biochemical logic of metabolism 63
Discussion 73
Bibliography 89
Declaration 110
3
Acknowledgments
I would like to thank Ron Milo, my advisor, for giving me the freedom to pursue my
research dreams and backing me for every scientific and personal decision. I could not
ask for a more supportive advisor.
I would like to thank Dan Tawfik for endless discussions and suggestions which
pushed my research forward and made it much more fun. I really appreciate the time
Dan invested in our interaction.
I also thank Naama Barkai for very helpful discussions that improved the quality of
my research.
In addition, I would like to thank everyone in the Milo lab for creating a wonderful
research environment. A special thanks to Elad Noor and Avi Flamholz, with whom I
did much of the work presented here.
Finally, I would like to thank my beautiful cat, Pakelet, who fills my days with
tenderness and joy. I dedicate this work to her.
4
Abbreviations
Fd Ferredoxin
LP Lipoprotein
MPT Methanopterin
PEP Phosphoenolpyruvate
Pi Inorganic phosphate
PPi Pyrophosphate
Rubisco Ribulose-1,5-bisphosphate carboxylase/oxygenase
THF Tetrahydrofolate
EMP Embden-Meyerhof-Parnas
TCA Tricarboxylic acids
kcat Enzymatic turnover number
KM Michaelis constant
kcat/KM Enzyme efficiency
KS Binding affinity
LogP Octanol/water partition coefficient
LogS Solubility in water
MW Molecular weight
Da Dalton
PSA Polar surface area
NPSA Non-polar surface area
NCA Number of charged atoms
HBI Hydrogen bond inventory
NRB Number of rotatable bonds
Q reaction quotient (mass-action ratio)
K Equilibrium constant
rG Transformed Gibbs energy of a reaction
rGo Transformed Gibbs energy of a reaction under reactant concentrations of 1M
rGm Transformed Gibbs energy of a reaction under reactant concentrations of 1mM
fG Transformed Gobbs energy of formation
Eo Transformed reduction potential
Central-CE Central-carbohydrates-energy
Central-ANF Central-amino-acids-fatty-acids-nucleotides
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Summary of Findings
I uncover several global trends that shape the global distribution of important
metabolic parameters. Evolutionary pressures and physicochemical constraints that
account for these trends are suggested. Analyzing the kinetic parameters of enzymes I
find that the average enzyme exhibits kcat and kcat/KM values much below the
characteristic textbook portrayal of kinetically superior enzymes. To account for these
findings, I find strong indications that maximal rates may not evolve in cases where
lower selection pressures are expected and that the physicochemical properties of
substrates constrain the optimization of kinetic parameters. I also find a general trend
in the concentrations of metabolites: in several organisms and growth conditions,
living cells minimize the concentrations of non-polar, un-charged metabolites. I
suggest that this can be attributed to an evolutionary pressure to avoid an unspecific
hydrophobic effect. These findings shed light on the evolution of the internal makeup
of living cells and can assist in establishing metabolic models that support synthetic
biology and metabolic engineering efforts.
I established quantitative frameworks for the analysis of metabolic pathways
according to their kinetic and thermodynamic features. Specifically, using a novel
estimation criterion of a pathways kinetics the pathway specific activity I
evaluate and compare various alternative carbon fixation pathways, in spite of kinetic
data scarcity. Using two thermodynamic frameworks, one treating metabolic
pathways as black-boxes while the other addresses their structure directly, I uncover
general constraints imposed on the environments in which pathways can operate, on
their general structure and on the cellular resources they consume. Using this
approach, I analyze and explain the structure of natural carbon fixation pathways as
well as the structure of glycolysis.
Finally, by exploring the space of carbon fixation pathways that can be
assembled from all ~5000 metabolic enzymes known in nature, I suggest a new
family of synthetic carbon fixation pathways that utilize the most effective
carboxylating enzyme, PEP carboxylase. One such cycle, which is predicted to be 2-3
times faster than the reductive pentose phosphate cycle, utilizes the core of the
naturally evolved C4 cycle and offers an exciting avenue for exploration in the grand
challenge of enhancing food and renewable fuel production via metabolic engineering
and synthetic biology.
6
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7
Introduction
The study of metabolism has been at the center of biological research for over one
hundred years 1. Yet, with the advent of molecular biology and genetic research, some
have misguidedly treated metabolism as a field to be mastered and then put aside 2.
Recent years, however, have witnessed a renaissance in metabolic research 3-7
.
Recently emerging grand challenges in sustainable energy, green chemistry and
pharmaceuticals have elevated the importance of metabolic engineering 8-11
. Deep
understanding of metabolic pathways is essential for such efforts; in order to redesign
metabolism one must gain a solid grasp of the biochemical principles governing it.
The research presented here focuses on several complementary aspects of
metabolism: (1) Understanding the evolutionary and physicochemical factors shaping
global metabolic networks; (2) Developing tools to analyze and compare metabolic
pathways; (3) Explaining the structures of pathways from basic biochemical
principles; (4) Designing novel synthetic pathways capable of achieving a giving
metabolic aim while satisfying biochemical constraints.
Of special importance are carbon fixation pathways. Many of the different
metabolic aspects listed above were developed or demonstrated using carbon fixation
pathways as model metabolic pathways.
Enzymatic parameters
A large body of literature discusses the complex interplay between the various
parameters of enzymatic catalysis 12-18
. Yet, the selective pressures that shaped these
parameters remain largely unclear. While traditionally kcat/KM was thought to be an
optimized quantity 13,17-19
, other alternatives were proposed 20-22
. For example, KM
values may have evolved to match physiological substrate concentrations 23
, whilst a
substrate-saturated enzyme is expected to maximize kcat and to be insensitive to KM
17,24.
There are also several known physicochemical constraints that set boundaries
to kinetic parameters 25,26
. For example, theoretical limitations suggest that kcat is
unlikely to be higher than 106-10
7 s
-1 15,27
. Furthermore, the apparent second-order rate
for a diffusion limited enzyme-catalyzed reaction with a single low molecular mass
substrate (kcat/KM) cannot exceed ~108-10
9 s
-1M
-1
28,29. The activation energy of the
reaction, as reflected in the un-catalyzed rate, also comprises a barrier: the enzymatic
8
acceleration of an extremely slow reaction, even by many orders of magnitude, may
still result in a relatively slow catalyzed rate 14
. The overall thermodynamics of a
reaction puts further limits. The Haldane relationship 30
states a dependency between
the kcat/KM of the forward (F) and backward (B) reactions, such that Keq=(kcat/KM)F /
(kcat/KM)B, where Keq is the reactions equilibrium constant. Therefore, even when
kcat/KM in the favorable direction is diffusion
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