PP1-Introduction to metabolism - Chem130B

8/7/2019 PP1-Introduction to metabolism - Chem130B

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Michael Sinensky, Ph.D.

[email protected]


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Prerequisites:A letter grade of C or better in Chem 112B, Chem 130A

and Biol 3. Chem 100W.Course Description and Goals

Chem 130B is the second semester of a three semester biochemistry

lecture course. Topics covered include the metabolism of carbohydrates,

lipids and amino acids; membrane transport; metabolic interactions and

control; and energy utilization. The metabolic basis of diseases involving

these processes will also be discussed.


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Student Learning Objectives

To become familiar with the major metabolic pathways ofcarbohydrate, lipids, amino acids and nucleotides with astrong emphasis on mammalian (human) systems.

To understand the major mechanisms governing the breakdown of nutrients to produce energy and to store it.

To understand the regulatory mechanisms governing thesepathways including genetic mechanisms.

To appreciate the role of metabolic dysfunction in disease.

To be able to read and understand the primary literature inintermediary metabolism.


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Outline of grading:

Midterm- multiple choice-~20%

In class 6x20 minute quizzes~ 20%

Final exam- multiple choice- ~25%

Term paper (10 pages) due by midnight on ~May17~35%- (to be submitted by e-mail)


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Introduction to metabolism:

Metabolism is the enzymatically catalyzed chemical conversion of

nutrients to biological end-products or of the degradation of those

products-essentially, metabolism is biological chemistry.

Catabolism- break down of nutrients or cell constituents to yield

energy, remove biologically harmful molecules or to salvage cellconstituent components.

Anabolism- Synthesis of complex molecules from precursors.


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ATP and NADPH are the sources of free energy for biosynthetic reactions.

(NAD+)


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Overview of aerobic catabolism:Dietary

nutrients are converted to common

endproducts for energy (and beginningmaterials for biosynthesis).


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NADH-redox in catabolism

ATP- biochemical activation

NADPH-redox in anabolism

3 key molecules of metabolism :

Living systems are all about energy:

How is it generated?

How is it stored?

How is it mobilized?

How is it utilized?

How are these processes regulated?

Glucose (hexoses) is the key extracellular source of NADH, ATP and,

indirectly, NADPH.


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the conversion of NAD+ to NADH

NADH is the product of mostcatabolic redox reactions- i.e. most

reducing power from catabolism is

first captured as NADH (FADH).

+2H+ + 2e-


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+

+ATP

+ADP

NAD kinase

+

+

Conversion of NAD+ to NADP+ by NAD kinase*- NADPH is the key source of

reducing power forbiosynthesis

NAD+ + ATP NADP+ + ADP

*A kinase is an enzyme that catalyzes

phosphoryl transfer from ATP to an acceptor,usually an alcohol.


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Ribose

Adenine

ATP is the immediate source of energy for most biochemical

reactions, as well as for thermogenesis. Storage of energy, including

that in ATP is the form of polysaccharides (i.e. glycogen) and fat (i.e.

triglyceride)


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Glycolysis- a

catabolic pathwaygenerates ATP

and NADH


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Synthesis of

Sphingomyelin

An example of the utilization

of ATP and NADPH to

synthesize a structural

component of cell

membranes: an anabolic

pathway.


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Methods for the elucidation of biochemical pathways:

Genetic manipulations- mutants, knockouts (siRNA) and transgenic cells

and organisms( particularly mice).

Isotopic tracers- usually radioactive but can use non-radioactive tracers in

conjunction with NMR or mass-spectrometry

Analytical methods to isolate the radioactive precursors and products with

enzymatic blocks-

Chromatographic techniques: HPLC, TLC, GLC etc.

Pulse-chase studies

Synthesis of precursors


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Alpha, Beta, and Gamma

Historically, the products ofradioactivity were called alpha,

beta, and gammawhen it was found that they could be

analyzed into three distinct species by either a magnetic

field or an electric field. -rays are He nuclei (mass=4,charge =+2), rays are high energy electrons, -rays areelectromagnetic radiation.

Beta rays are emitted from the nucleus of a radioactive

atom. Examples of beta emitters commonly used in

biological research are: hydrogen-3 (tritium), carbon-14,

phosphorus-32, phosphorus-33, and sulfur-35. -rays areionizing.

http://web.princeton.edu/sites/ehs/osradtraining/radiationproperties/radiationproperties.htm#Half-Life
http://hyperphysics.phy-astr.gsu.edu/hbase/nuclear/radact.htmlhttp://hyperphysics.phy-astr.gsu.edu/hbase/nuclear/radact.htmlhttp://hyperphysics.phy-astr.gsu.edu/hbase/nuclear/beta.htmlhttp://hyperphysics.phy-astr.gsu.edu/hbase/nuclear/radact2.htmlhttp://hyperphysics.phy-astr.gsu.edu/hbase/nuclear/radact2.htmlhttp://hyperphysics.phy-astr.gsu.edu/hbase/nuclear/beta.htmlhttp://hyperphysics.phy-astr.gsu.edu/hbase/nuclear/radact.htmlhttp://hyperphysics.phy-astr.gsu.edu/hbase/nuclear/radact.html


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Technology of liquid scintillation counting

Radioactive isotopes have characteristic

half-lives.

Random first-order process.t = ln2/k = 0.693/k ; k=rate constant


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Technology of liquid scintillation counting

The scintillation solvent in most

common use is toluene.

Crystalline scintillation systems

are also sometimes used.


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Radio HPLC demonstration that lanosterol is a precursor of cholesterol- the AR45

mutant is a cholesterol auxotroph of the CHO cell- cells are incubated with 14 CH3COOH.

Lanosterol accumulates to high levels-


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Chol.

Note lanosterol is converted to

cholesterol in wild-type cells but

not in the cholesterol auxotroph

215.


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Voet

Bio

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2004John

Wiley&Sons,Inc

.

Pathway of arginine biosynthesis as indicated by nutritional

status of various genetic blocks

(Complementary arginine auxotrophs ofNeurospora crassa).

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561

Grows on

arginine,

ornithine

orcitrulline

Grows on

arginine,citrulline

Grows

only onarginine,


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An E. colimutant blocked in shikimic acid synthesis was originally

isolated as an auxotroph for a mix of aromatic amino acids. The growth

requirement could be met by shikimic acid and labeled shikimate could

later be shown to be incorporated into aromatic amino acids.

Metabolic mutants have been used extensively to define metabolic pathways.


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Voet

Bio

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2004John

Wiley&Sons,Inc

.

Figure 16-13 Pathway for phenylalanine degradation.

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560

Caution- accumulation of a

compound in a mutant doesnot necessarily make it an

intermediate. Not likely to be

a problem because of the

sophistication of

contemporary genetics


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The flow of a pulse of radioactivity from precursor to product.

Label = acetate

A= mevalonic acid

B= squalene

Product =

cholesterol

n.b. In reality

things are never

this clean. Many

technical

problems.

Specific radioactivity= dpm/mole


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In this example from the textbook the vast majority ofevidence favoring scheme II came from directly

demonstrating conversion of the possible precursor (ether

or vinyl ether) to product using labled precursor.

This reaction

cant be

demonstrated invitro.

+ R*OH

Which is precursorand which is

product?


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Some elements in the demonstration that AB in a biochemical pathway

Genetic or drug inhibition of reaction in organism results in accumulation ofA.

Relief of block results in AB

The moles of B formed are equivalent to the moles of A disappearing.

In vitro verification that AB occurs by a particular enzymatic process.

Structural verification that accumulated A is what it is thought to be.

Precursor product analysis by metabolic block: the conversion of A to A


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t

Precursor-product analysis by metabolic block: the conversion of A0to A

requires an intermediate derived from mevalonate.

Mevalonate synthesis inhibitor Mevalonate synthesis

defective mutant


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Detection of metabolic conversion to product in a crude cell free extract:

analysis by thin layer chromatrography; detection by autoradiography or

fluorography.

Quantitation by liquid scintillation counting

Characterization of prelamin A endoprotease or Zmpste24


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Vmax =kEt


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Figure 7-8b The generation of the gas phase ions required forthe massspectrometric analysis of proteins. (b) By matrix-assisted laser

desorption/ionization (MALDI).

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72


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In the rare genetic disease, restrictive dermopathy, Zmpste24 is missing.

Patients cells accumulate the metabolite shown which is the carboxyl

terminal peptide of the protein (tryptic digest) ; characterization by MALDI-

TOF mass-spectrometry.


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Molecular techniques for studying the regulation of gene expression


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Control and coding elements of a eukaryotic gene


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Assembly of the preinitiation complex (PIC) on a TATA boxcontaining

promoter. This can be done in vitro on a naked DNA template.

RNAPII= RNA

Polymerase II;

The entire PIC is

often referred as

RNAPII

holoenzyme,

emphasizing the

functionality ofthe entire

multisubunit

complex.

orInr


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Chromatin is a nucleoprotein: notnaked DNA. It needs

to be remodeled in order to permit transcription to occur.

Chromatin Remodeling


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g

Complex

Binding

Complex

Binding

Loosening of the

Chromatin Structure

Loosening of the

Chromatin Structure

RemodelingRemodeling

Octamer TransferOctamer Transfer

Octamer SlidingOctamer Sliding

NucleusNucleus

ATP

ADP

DNA-Chromatin ComplexDNA-Chromatin Complex

Swi/SNF

Complex

Swi/SNF

Complex

Swi/SNF

Complex

BRG1

BRG1

Swi/SNF

Complex

BRG1

BRG1

2009ProteinLounge.com

2009ProteinLounge.com

C


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Regulated transcription:

The transcription factor

SBF recruits the co-activator Mediator

which facilitates the

formation of the

RNAPII holoenzyme at

the promoter.

Chromatin

remodeling


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Overview of the assembly of the transcription complex and the associated proteins-the

CTD (carboxylterminal domain) of RNAPII is especially important in interactions

regulating RNAPII activity.


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Fully assembled transcription complex (TAF= TBP- associated

factors)

General transcription

factors

Upstream

transcription

factors

recognize

specificupstream

regulatory

sequences.


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Enhancers and activators-

activators may be bound

constitutively to ubiquitousenhancer sequences or may

exhibit regulated and highly

gene specific activation. HMG

proteins produce DNA loops

that allow regulatory

sequences distant from the Inr

to affect RNAPII.

Eukaryotic promoters and regulatory proteins. RNA polymerase II and its associated


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basal (general) transcription factors form a preinitiation complex at the TATA box and Inr

site of the cognate promoters, a process modulated by transcription activators, acting

through mediator.


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Steroid hormone regulated transcription factors have in addition to the

activation domain (AD) and DNA binding domain (DBD) common to all TAFs a

ligand binding domain which is the site of hormonal regulation


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Microinjection of DNA into the pronucleus of a fertilized mouse ovum.


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Carriers for genetic modification/gene transfer/gene therapy


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There are radiological techniques that allow the visualization and

quantitation of metabolites in whole animals. Example: the expression of

creatine kinase results in accumulation of phospho creatine in transgenic

mouse liver as demonstrated by localized in vivo 31 P NMR.


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SDS-PAGE

Immuno (Western) blot detection of

protein expression

Chromatin immunoprecipitation (ChIP): analysis of

C


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DNA sequences recognized by the CREB

transcription factor.

CREBMechanical shear

Polymerase Chain reaction


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Polymerase Chain reaction

e.g.Anneal @50-60C; extend

@72, melt @95


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Promoter analysis utilizes reporter assays for an easy to assay gene product.

Luciferase can be assayed in a luminometer which quantitates light emitted by

cleavage of the enzymes substrate luciferin.

Fi 16 17 Th t b li i i f th it t i


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oet

Bio

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istr

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2004John

Wiley&Sons,In

c.

Figure 16-17 The metabolic origin of the nitrogen atoms in

heme.Use of mass isotopes.

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PP1-Introduction to metabolism - Chem130B