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Lecture 15 1
Introduction to Metabolism &
Experimental Techniques
What does “metabolism” mean?
How are metabolic processes organized?
Techniques for studying metabolism
Reading assignment:
P. 485-
p. 90 & 91 (IEF & 2DE)
Chapter 9.3 (p. 324-329)
Lecture 15 2
What is metabolism?
Sum of chemical transformations occurring in
living cells /organisms
Conversion of nutrients into chemical energy
for energy-requiring activities and for
temporary storage
Essential to maintain life
Lecture 15 3
Metabolism consists of two
contrasting processes
Adapted from (^) Lehninger 5th ed., Principles of Biochemistry, Cell macromolecules Proteins Polysaccharides Lipids Nucleic acids Precursor molecules Amino acids Sugars Fatty acids Nitrogenous bases Anabolism (reductive, endergonic) Energy depleted end products CO 2 H 2 O NH 3 Energy-yielding nutrients Carbohydrates Fats Proteins Catabolism (oxidative, exergonic)
ADP,
NAD+, FAD
NADP+
ATP, FADH 2
NADH,
NADPH
Chemical energy Lecture 15 4
Metabolic reactions are organized
into pathways
Fig. 15-
Lecture 15 5
Enzymes can associate to from
multi-enzyme systems
Cell membrane Cytosol Lecture 15 6
Anabolic and catabolic pathways
are reciprocally regulated
Anabolism and catabolism occur simultaneously
in the cell.
Biosynthetic and degradative reactions are
regulated separately
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Lecture 15 7
Organisms need to maintain a
steady-state level of metabolites
Steady state (homeostasis)
= essentially constant composition and
concentration of metabolites
⇓ to keep the [metabolite] +/- constant:
Metabolite flow rate (flux) can be regulated by
Lecture 15 8
Enzyme levels and activities vary
Proteomics: 2D-gel electrophoresis
2nd dimension: SDS-PAGE
Figure 3-
Spot excision and detection
by mass spectrometry
Lecture 15 14
Metabolomics: GC-MS
Metabolites extracted from cell lysate Detector Lecture 15 15
NMR spectroscopy
Rest, active, recover Kushmerick PCr Pi
ATP
~P changes by 31 P NMR spectroscopy:
Exercising human forearm
Lecture 15 16
Radioisotope tracers
Extend the limits of detection
From μM or nM to pM or fM!
Act as a tag
Can be used to elucidate metabolic sequences.
Example for a Pulse-Chase experiments:
Feed cells a labeled substrate for a short time (pulse-phase). Introduce excess of unlabeled substrate for prolonged time (chase-phase). Take samples at time points, separate components (e.g. on SDS-PAGE) and measure radioactivity. -> follow the fate of substrate and subsequent intermediates.
Lecture 15 17
Example of a pulse-chase
experiment:
Lecture 15 18
Pulse-chase question
How would you interpret these results?
Lecture 15 19
Inhibitors
Helpful in determining the
physiological role of enzymes.
Take an inhibitor
that is known to inhibit
the purified enzyme X.
-> Add inhibitor to cell
to study its effects.
Disadvantage:
Inhibitor may
not be specific.
A -> B -> C -> D? or D -> C -> B -> A?
inhibitor
Lecture 15 20
Mutants
Isolate mutant in specific enzyme and study
the effect on the cell.
Specific
Very powerful: This technique has been
used to elucidate whole pathways in
microorganisms.
Example: Beadle and Tatum and arginine
metabolism.
