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.

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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