1. Welcome to BCH3120: General Intermediary Metabolism
Slides for Lectures 1 and 2
Welcome to BCH3120: General Intermediary Metabolism
Mondays 10:00-11:30am Wednesdays 08:30- 10:00am
Marion Auditorium
Dr. Mary-Ellen Harper (Lecturer, Course Co-ordinator)
Dr. Amanda MacFarlane (Lecturer)
Dr. Zemin Yao (Lecturer)
TAs: Brittany Beauchamp and Ariana Noel

2. Honours Project Networking Event
When: Friday, January 13th, 2012 from 3:00 to 6:00pm
Where: Lobby of Biosciences Complex
Purpose: To help you find a project supervisor, the program is organizing a poster session/networking event, presented by its professors.  Here you will have an opportunity to meet some of the department’s researchers and learn about the work they do.  This is an excellent networking opportunity – several of our current 4th year students found a supervisor after attending this event.
I will be out of the country that day – if you are interested in a fourth year project in my lab (bioenergeticslab.com) please me and describe why you are interested and describe any lab experience…

4. Effects of exercise
and training
Effects on metabolism of eating,
fasting, and starvation
Major Metabolic
Diseases
- Types 1 and 2
Diabetes

6. Manga Guide to Biochemistry
Takemura, Kikuyaro and Sawa
2011, Ohmsha Press

7. Intermittent Continuous
Energy Intake
(Food)
Energy Content of the Body
Energy Expenditure
Avg. N.American
Pro. 12%
Pro. 25,000 kcal
(15% of body E)
Fat %
Fat 141,000 kcal
(84% of body E)
CHO 48-58%
CHO 1,800 kcal
(1% of body E)
Circulating:
Glu 80 kcal
FFA 3 kcal
TG 30 kcal
ATP content of body: 0.7 kcal
Importance of dietary CHO intake, and stored body fat
ENERGY CONTENT is only a small proportion of ENERGY FLOW

8. A plethora of pathways!

11. Metabolism
the sum of biochemical processes involved in the synthesis, breakdown, and inter-conversion of constituents in cells and organisms
Intermediary metabolism is where anabolism and catabolism intersect, often sharing pathways and metabolites.
Because the pathways of intermediary metabolism are essential for life, they are tightly regulated through a variety of mechanisms.

12. Heterotrophs:
require complex nutrient molecules, like glucose, as a source of energy.
Autotrophs:
can synthesize complex molecules from simple precursors like CO2 and ammonia

14. or Body Stores
of Energy

15. Overview of interrelationships between major anabolic and catabolic pathways.

16. Learning is easiest, when the information is perceived as relevant and/or interesting…
Is intermediary metabolism relevant in the post-genomic era?
How might you use the information from BCH3120?
‘Personal - Practical’: Effects of diet, exercise, disease on characteristics of energy metabolism (body composition, types of fuels oxidized…)
‘Metabolism in the News’ and integrated examples in lectures
‘Medical Implications’ examples
‘Current Research’ lectures, and relevant abstracts included in lectures

17. Introduction to Fundamental Concepts
Organisation of BCH3120
Purpose
Content
Evaluation
Introduction to Fundamental Concepts

18. Purpose of BCH3120:
An integrated understanding of the major pathways of carbohydrate, lipid and amino acid metabolism

19. BCH3120 General Intermediary Metabolism
Organisation of the course
Purpose
Content
Evaluation
Introduction
Few basic concepts

20. BCH 3120 Content (Continued)
Introduction and Principles of Metabolism (Lectures 1, 2: Jan 9, 11) Dr Harper
Carbohydrate Metabolism
Glycolysis, Fermentation and the Pentose Phosphate Pathway (Lectures 3 and 4; Jan 16 and Jan 18) Dr. Harper
Gluconeogenesis, Glycogenolysis and Glycogen synthesis (Lecture 5; Jan 23) Dr. Harper
Tricarboxylic acid (TCA) cycle (Lecture 6; Jan 25) Dr. Harper
Oxidative Phosphorylation (Electron transport chain and ATP synthase) (Lecture 7; Jan 30) Dr Harper
Research lecture: Costs and Benefits of Mitochondrial Uncoupling (Lecture 8; Feb 1) Dr Harper
Midterm 1 (Feb 6)

21. BCH 3120 Content (Continued)
Metabolic Control Analysis & Introduction to Metabolomics (Lecture 9; Feb 8) Dr Harper
Lipid Metabolism. Dr Yao
Fatty acid catabolism (Lecture 10; Feb 13)
Fatty acid and triglyceride synthesis (Lecture 11; Feb 15)
Phospholipid metabolism (Lecture 12; Feb 27)
Biosynthesis of cholesterol (Lecture 13, Feb 29)
Introduction to lipoprotein metabolism (Lecture 14, March 5)
Regulation of cholesterol homeostasis (Lecture 15, March 7)
Amino Acid Metabolism- Dr Yao
Amino acid deamination and the urea cycle (Lecture 16, March 12)
Biosynthesis of amino acids and related molecules (Lecture 17; March 14)
Regulation of amino acid metabolism (Lecture 18; March 19)
Midterm 2 (March 21)

22. BCH 3120 Content (Continued)
Purine and Pyrimidine Metabolism - Dr. MacFarlane
Folate and ‘1-carbon’ metabolism and the ‘methyl-folate trap’ (Lecture 19; March 26)
Pyrimidine biosynthesis, and cancer chemotherapeutic agents (Lecture 20; March 28)
Metabolism: Toward an Integrated Understanding – Dr. Harper
Diabetes and Intermediary Metabolism (Lecture 21; April 2)
Exercise and Intermediary Metabolism (Lecture 22; April 4)
Course review class (Lecture 23; April 9) Dr. Harper

23. BCH 3120 Content Prerequisite: BCH2333 or BCH2733
If you do not have this prerequisite you must obtain permission
from the Department (contact Dr. Harper, provide reason and transcript).
Lecture notes: Slide sets posted at least 24h prior to each lecture.
Textbooks:
Biochemistry, Third or Fourth Edition
D. Voet and JG. Voet
Available at: University Bookstore
Lehninger: Principles of Biochemistry, Fourth or Fifth Edition
Available at the University Bookstore and the Agora Bookstore

24. Lecture notes: Will consist of slide sets, which will be posted
at the course website 24h prior to each lecture.
On occasion, relevant papers from the literature will be assigned for reading. The professor will tell you what information in the papers will be required for the purposes of exams.

25. Cornerstone of intermediary metabolism
Tricarboxylic Acid Cycle
Cornerstone of intermediary metabolism
– MEMORIZE ASAP
Fig. 26-1

26. with prizes… Don’t miss it!
Dr. Harper prior to Jan 30th if you will submit an entry.
SONG CONTEST!
with prizes… Don’t miss it!
End of class on Feb.1st

28. BCH3120 General Intermediary Metabolism
Organisation of the course
Purpose
Content
Evaluation
Introduction
Few basic concepts

29. BCH3120 Evaluation Intended to assess your: Fundamental comprehension
Ability to integrate and apply the information

30. BCH3120 Evaluation

31. BCH3120 Evaluation Mid-term exams
Focus on new/untested material (e.g., MT#2 will focus on course material AFTER MT#1)
Duration: 60 minutes
Format:
Multiple choice questions
True or false questions
Tables or pathways to complete (« fill in the blanks »)
** In this room; we will have ~8 proctors to make this possible. Leave your coats and notes in your locker/elsewhere if possible!

32. BCH3120 Evaluation Final Exam Cumulative Worth 45% of final mark
Increased emphasis on cross pathway interactions/ metabolic networking. i.e., your integrated understanding of the material
Format: similar to that of the mid-term exams
**If your mark in the final exam is better than the average of your midterm marks, then the final exam mark will become your final course mark.

33. Policy on Exams:
If you miss a mid-term exam due to illness, obtain a doctor’s note and forward it within 48h by Mme. Johanne Bouchard (Room 4170, Roger Guindon Hall) for departmental approval.
Without a valid note from a doctor, you will get zero.
With an approved note, your final mark will be based on the performance overall in the exams completed over the term.
If you miss the final exam due to illness, contact the Faculty of Sciences office immediately to inquire about current Faculty policies and a possible deferred exam. If this is not approved by Faculty a mark of zero will be assigned for the final exam.

34. Questions about lecture materials:
one of the two Teaching Assistants,
Brittany Beauchamp (for students with last names starting with the letters A – M) or,
Ariana Noel (last names starting with N – Z)
Please be very specific, explaining what you do and don’t understand. Don’t leave Qs to the last hours prior to exams!!!

35. How might you use information from BCH3120?
Learning is easiest, when the information is perceived as relevant and/or interesting…
How might you use information from BCH3120?

36. Relevance to future potential careers in medical research (e. g
Relevance to future potential careers in medical research (e.g., cell biology)
intermembrane
space
matrix
inner
membrane
outer membrane
E.g., Role of metabolic
Processes in
Mitochondria in the
Development of
Neurodegenerative
Diseases.

37. Relevance to future potential careers in industry
(e.g., biotech or pharmaceutical)

38. Relevance to a better personal understanding of advances in medicine and life sciences
To know more about:
Different types of diabetes
Complex origins of obesity
Genetic metabolic diseases
Technologies used in these and other areas of metabolic research (e.g., PET scans)
Crawford et al, 2010

39. Relevance to a better personal understanding of Nutrition and Health
Energy,
Nitrogen
Nutrients
Health
pages/foodland.html
trailrunning06.html

40. BCH3120 Metabolism in the News
An anti-aging method that works in virtually all species studied thus far…
Caloric Restriction: but HOW does it work/ What are the mechanisms?
What are the risks? Are their pharmaceuticals that can replicate the
beneficial effects?

41. BCH3120 General Intermediary Metabolism
Organisation of the course
Purpose
Contents
Evaluation
Introduction
Fundamental concepts
TCA Cycle Song Contest: Wed Feb 1
MT#1: Mon Feb 6
MT#2: Wed March 21
Today
Experimental approaches
General principles of Regulatory Control of Metabolic Pathways

42. Studying Metabolism in the Post Genomic Era
‘Big Science’
Gene sequencing and Genome databases
DNA microarrays: mRNA expression levels and microRNAs
Proteomics: protein expression levels, and post-translational modifications
Metabolomics: comprehensive analyses of metabolite levels

43. Genome databases
Sequence and map data from the whole genomes of over 1000 organisms. All three main domains of life (bacteria, archaea, and eukaryota) are represented, as well as many viruses, phages, viroids, plasmids, and organelles. [take a look at the NCBI website!]
This incredible source of information should be considered when studying intermediary metabolism.
An understanding of metabolic pathways and their control is a great advantage in researching genes of interest in genomic research.
What is the metabolic impact of a mutation (‘variant’) in a specific gene?
Gene sequence – variants?
mRNA expression levels ?
Protein levels and characteristics ?
Metabolite levels ?

44. DNA microarrays
Purpose: To simultaneously assess expression levels of thousands of genes. Check out, for example, the website of Affymetrix, which produces gene chip arrayshttp://
The PrimeView™ Human Gene Expression Array enables you to:Measure gene expression of more than 36,000 transcripts and variants per sample
To assess the origins and development of disease, the subtypes of disease; disease prognosis (e.g., in cancer); treatment outcomes… (E.g., Ottawa Hospital Weight Management Clinic: Thousands of patients followed; Rate of weight loss and risk for diabetes etc. varies greatly and is related to unique gene expression profiles)
To clarify the specificity of pharmaceuticals. (E.g., hypo-cholesterolemic drugs, which represent > $20 billion market internationally. Microarray studies have demonstrated that some drugs affect metabolic pathways beyond those that were initially targeted.)

45. ‘Cluster Analysis’
Many systems biology approaches for data analysis. Cluster-type
approaches assess levels of transcripts, proteins or metabolites
in relation to known pathways and biological functions.

46. Gene sequence – variants?
mRNA expression levels ?
Protein levels and characteristics ?
Metabolite levels ?
Proteomics
Purpose: expression levels and characteristics of proteins E.g., within cells and tissues of an organism.
Proteomic approaches allow investigation into responses of a biological system to different stimuli, e,g, disease states, or following drug treatment.
Recent progress in analytical techniques for the study of proteins has led to significant improvements in the ability to identify peptides and their posttranslational modifications on a high throughput basis.
E.g., “A novel proteomic approach for the discovery of chromatin associated protein networks” (By JP Lambert, L. Mitchell, A Rudner, K Baetz and D Figeys) Mol Cell Proteomics Dec 22. [Epub ahead of print]. **From our Ottawa Institute of Systems Biology.

47. The complexity of an organism is the result to only a small extent of gene expression alone. The one gene-one protein concept is incorrect. The 2002 Nobel Prize in chemistry was awarded to Drs Fenn, Tanaka and Wuthrich for their contribution to the development of mass spectrometric and NMR methods for biomolecules, especially proteins.
Figures modified from Am J Physiol Lung 2004: 287:L1 and Nobel e-Museum.
About 20, ,000 protein encoding genes

48. Metabolomics
Gene sequence – variants?
mRNA expression levels ?
Protein levels and characteristics ?
Metabolite levels ?
A new investigative field that applies advanced analytical techniques to study metabolite profiles in serum or tissue samples.
Several hundreds of metabolites simultaneously!
By comparing metabolite levels in sequential samples and inferring metabolite flux, metabolomics can also identify rate-controlling steps – e.g., that could be targeted for the development of new drugs.

49. Example: Metabolite profiles of brain of individuals with schizophrenia vs healthy controls
The results strongly suggest that markers of oxidative stress and mitochondrial perturbations provide a pathological signature for the disease.
Figures obtained from Mol Psychiatry 2004.

50. An integrated understanding of the pathways of carbohydrate, lipid and amino acid metabolism
… IS FUNDAMENTALLY IMPORTANT!

51. Useful Websites: Interactive Metabolic Pathways
The ExPASy (Expert Protein Analysis System) proteomics server of the Swiss Institute of Bioinformatics (SIB):
IUBMB-Nicholson Metabolic Maps, Minimaps & Animaps:

52. Example: IUBMB-Nicholson Metabolic Maps

53. Cell-cell communication & compartmentalisation Thermodynamics
Cell biological and Biochemical Approaches to Assess Regulation and Control of Metabolic Pathways
Key Concepts:
Cell-cell communication & compartmentalisation
Thermodynamics
Inhibition and allostery
Post-translational modifications
Enzyme turnover

54. Cell-cell communication & compartmentalisation
Procaryote – unicellular organism; cells lack membrane bound nuclei; e.g., bacteria, blue-green algae and mycoplasma

55. Cell-cell communication & compartmentalisation
Eukaryote – cell or organism having membrane-bound, structurally discrete nucleus other well-developed subcellular compartments Includes all organisms except viruses, bacteria, & cyanobacteria (blue-green algae)

56. Cell-cell communication & compartmentalisation
Localization of distinct metabolic pathways: Examples

57. Cell-cell communication Direct connection: Gap Junctions
Connexins: key mediators of endocrine function.
Bosco D, Haefliger JA, Meda P. Physiol Rev Oct;91(4):
Department of Surgery, University of Geneva Medical School, Geneva, Switzerland.
Abstract
The appearance of multicellular organisms imposed the development of several mechanisms for cell-to-cell communication, whereby different types of cells coordinate their function. Some of these mechanisms depend on the intercellular diffusion of signal molecules in the extracellular spaces, whereas others require cell-to-cell contact. Among the latter mechanisms, those provided by the proteins of the connexin family are widespread in most tissues. Connexin signaling is achieved via direct exchanges of cytosolic molecules between adjacent cells at gap junctions, for cell-to-cell coupling, and possibly also involves the formation of membrane "hemi-channels," for the extracellular release of cytosolic signals, direct interactions between connexins and other cell proteins, and coordinated influence on the expression of multiple genes. Connexin signaling appears to be an obligatory attribute of all multicellular exocrine and endocrine glands. Specifically, the experimental evidence we review here points to a direct participation of the Cx36 isoform in the function of the insulin-producing β-cells of the endocrine pancreas, and of the Cx40 isoform in the function of the renin-producing juxtaglomerular epithelioid cells of the kidney cortex.

58. Cellular compartmentalisation
Thermodynamics
Inhibition and allostery
Post-translational modifications
Enzyme turnover A B C D 1 2 3
Thermodynamics
The committing step catalyses a reaction that has a large free energy change (irreversible step)
The other enzyme reactions are characterized by a relatively lower free energy change

59. The Thermodynamics of Entropy: The Advantages of Being Disorganized…
The Oxidation of Glucose:
small number of relatively complex/ordered molecules to
a larger number of simpler molecules (increased entropy)
energy released !

60. Creating Order Costs Energy, but Order has Potential
(e.g., energy; information)
“There is a tide in the affairs of men,
Which, taken at the flood, leads on to fortune;
Omitted, all the voyage of their life
Is bound in shallows and in miseries.”
Shakespeare Julius Caesar, Act IV, Scene 3

61. Energy released from an EXERGONIC PROCESS
Can drive an ENDERGONIC PROCESS

62. Chemical example of ENERGY COUPLING:
ATP hydrolysis drives glucose phosphorylation

63. Enzymes catalyze reactions by lowering
the activation energy of the reaction process

64. Central Role of ATP in Metabolism
ATP is:
The chemical intermediate linking
energy-releasing
catabolic (exergonic) reactions
with
energy demanding
anabolic (endergonic) reactions

65. A B C D Thermodynamics and Control of Metabolic Pathways Free energy
Committing/Controlling step (1) A B C D 1 2 3
Free
energy
3rd enzyme reaction
2nd enzyme reaction
Carbon flux progression

66. Regulatory Control of Metabolic Pathways by Inhibition and Allostery
Cellular compartmentalisation
Thermodynamics
Inhibition and allostery
Post-translational modifications
Enzyme turnover
Regulatory Control of Metabolic Pathways by Inhibition and Allostery
Inhibition:
Common
Non-reactant molecule that resembles the substrate interferes with access
of the normal substrate to the substrate binding pocket
Endogenous inhibitor (end-products: “feedback inhibition” “negative effector”)
Exogenous inhibitor (poisons)

67. E1 = Threonine dehydratase
E1 is specifically inhibited allosterically by the end-product,
but by none of the four intermediates (B, C, D, or E).

68. Inhibition and Allostery
Binding of a ligand at one site affects binding of another at another site
Structural alterations interfere with the accessibility of other ligands, substrates or cofactors, or may disturb the catalytic site.
‘Allosteric’ enzymes exist in at least two different states (controlled by the regulatory substances):
R state (R= relaxed): high affinity for substrate
T state (T = tense): low or no affinity for substrate
Allosteric activators and inhibitors induce conversion from the
T to the R state, and the R to the T state, respectively.

69. Allosteric control of glycerol kinase: It’s smart/rational !
Background:
Both glycerol and glucose can replenish the Krebs cycle.
A ‘smart’ process/pathway:
The minor glycerol-dependent pathway is turned off when the usual substrate, glucose, is available.
Molecular mechanism:
… 2nd next slide

70. Allosteric control of glycerol kinase by fructose 1,6-bisphosphate (FBP)
Molecular details corroborate the allosteric control of glycerol kinase : fructose 1,6-bisphosphate locks GK in its TETRAMERIC, INACTIVE state.

71. Control by Post-Translational Modifications
An important additional means of control
i.e., beyond the ‘upstream’ control mechanisms such as:
Transcriptional control: controlling level of mRNA expression e.g., PPARs and their PPREs
Translational control: at initiation, elongation and termination steps in conversion of mRNA to protein

72. Post-translational Control
To modulate the activities of enzymes already synthesized
For acute control of enzyme activities (milli-seconds to minutes)

73. Post-Translational Modifications: Covalent Modifications
E.g., Phosphorylation by PKs
Other covalent modifications:
- Ca++
- Methyl
Acetyl
- glutathionylation
Induce conformational
changes, turning active site
ON or OFF
Reversible

74. Antioxid Redox Signal. 2008 Nov;10(11):1941-88.
Molecular mechanisms and clinical implications of reversible protein S-glutathionylation. Mieyal JJ, Gallogly MM, Qanungo S, Sabens EA, Shelton MD.
Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio
Abstract Sulfhydryl chemistry plays a vital role in normal biology and in defense of cells against oxidants, free radicals, and electrophiles. Modification of critical cysteine residues is an important mechanism of signal transduction, and perturbation of thiol-disulfide homeostasis is an important consequence of many diseases. A prevalent form of cysteine modification is reversible formation of protein mixed disulfides (protein-SSG) with glutathione (GSH). The abundance of GSH in cells and the ready conversion of sulfenic acids and S-nitroso derivatives to S-glutathione mixed disulfides suggests that reversible S-glutathionylation may be a common feature of redox signal transduction and regulation of the activities of redox sensitive thiol-proteins. The glutaredoxin enzyme has served as a focal point and important tool for evolution of this regulatory mechanism, because it is a specific and efficient catalyst of protein-SSG deglutathionylation. However, mechanisms of control of intracellular Grx activity in response to various stimuli are not well understood, and delineation of specific mechanisms and enzyme(s) involved in formation of protein-SSG intermediates requires further attention. A large number of proteins have been identified as potentially regulated by reversible S-glutathionylation, but only a few studies have documented glutathionylation-dependent changes in activity of specific proteins in a physiological context. Oxidative stress is a hallmark of many diseases which may interrupt or divert normal redox signaling and perturb protein-thiol homeostasis. Examples involving changes in S-glutathionylation of specific proteins are discussed in the context of diabetes, cardiovascular and lung diseases, cancer, and neurodegenerative diseases.
PMID: [PubMed - indexed for MEDLINE]

75. Control by Enzyme Turnover
The ubiquitin pathway is an important component for the
control of enzyme turnover
The PEST motif is a tag for the ubiquitinization of proteins:
Proteins with a N-terminal PEST motif “survive” for about 2-3 min
Proteins lacking a N-terminal PEST motif have half-lives of about 10 hours

76. Later this term, we will review METABOLIC CONTROL THEORY
We will see that Metabolic Control Analysis is an approach that allows researchers to identify the major controlling steps.
It is no longer acceptable to say that there is one rate limiting step in a metabolic pathway
Controlling steps can change based on changes in metabolic conditions, presence/absence of disease, etc.

77. Review questions
List some of the different major pathways related to intermediary metabolism.
Why are metabolic controls necessary?
What are some different types of regulatory controls of enzyme activity?
List some ways that results of DNA microarrays and proteomic arrays
could be of use in metabolic studies
What is metabolomics? Why is it useful?
What is a Metabolic Control Analysis?