Download 1 Confusion from last week: Purines and Pyrimidines

BIO 5099: Molecular Biology
for Computer Scientists (et al)
Lecture 10: Energy & Enzymes
http://compbio.uchsc.edu/hunter/bio5099
[email protected]
Confusion from last week:
“Small” RNAs are RNAs not involved in protein
production, e.g. snRNA
– Ribosomal rRNA is part of the machinery that produces
proteins (more in a moment)
First, a distinction without a difference. “Small RNA”
is somewhat ambiguous term
– Fine to say that rRNA is not a small RNA
Second, mRNA and tRNA are representations of
protein itself, where rRNA is not, hence the
distinction...
Purines and Pyrimidines
Pyrimidines are single dinitrogen heterocycles
Purines are two fused dinitrogen heterocycles
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Back to the Ribosome
Recall the Ribosome is the location of protein
synthesis, where mRNA is translated.
Today, we finish
up by looking
at tRNAs and
the other
molecules of
the complex.
tRNA function
Transfer RNA (tRNA) translates individual codons to
their corresponding amino acids.
One end has an anti-codon which binds to the mRNA.
The tRNA codon sequence is the same as the gene
sequence
– mRNA is inverse of DNA, tRNA is inverse of mRNA
– RNA, so U instead of T
Other end binds the appropriate amino acid.
– Specifically, but not too strongly (has to release it!)
tRNA structure
tRNA has a fixed conformation
(contrast with “floppy” mRNA )
RNA secondary structure is how nucleotides in the
RNA bind to other nucleotides in it
RNA tertiary structure is the complete 3D
conformation.
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Other parts of the Ribosome
Initiation factors:
– Proteins which start the process of translating mRNA
Elongation factors:
– Proteins which ensure that the translation proceeds
appropriately.
– E.g. Peptidase, which creates the peptide bonds
Termination factors:
– Detect STOP codons, and end the translation
Example biomolecular study
Ribosome is a good example of a biomolecular
machine
– Complex components, intricately assembled
– Key function: the mechanism for creation of all proteins
– Need to understand
•
•
•
•
All of its components (their structures and functions)
Their interactions
Energetics (which we ignored so far).
Synthesis, distribution, control, degradation....
Hierarchical study of structure and function
Core Functions of life
Energy: Capture, storage and use of energy
Anabolism: Creation of biomolecules
– Protein (Central Dogma, ribosomes, tRNAs)
– DNA duplication and transcription
– Lipids and energy storage molecules
Catabolism: Breakdown of inputs
– Getting energy from food, sunlight or redox potential
– Creating material building blocks
Control: Sensing and acting on state
(of the self & of the environment)
Reproduction: Making offspring
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Molecular Power
Adequate -G must be coupled to reactions that don't
occur spontaneously (most of biology).
– Too little energy, and necessary reactions don't occur
– Too much energy, and bonds inside important molecules (e.g.
proteins) can be disrupted, doing damage.
Also, may need other forms of energy (redox)
Where does the energy come from? How is it
delivered when and where it is needed?
Energy metabolism
ATP: powering life
Energy is obtained by the breakdown of food
(or photosynthesis, or redox reactions)
Energy is used by nearly all living processes
Adenosine Triphosphate (ATP) is the main energycarrying molecule of life
ATP
Adenosine is the same as the nucleotide in RNA.
Triphosphate is the addition of 3 PO4s
Phophate bonds are “high energy” in that
they release a
lot of energy
under hydrolysis
Hydrolysis
produces -G
7.3 Kc/mol
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Amazing ATP
Use of ATP is huge.
– A cell uses about 12nM ATP/day (7.5X1015 molecules)
– For a “basal” human, about 125 moles of ATP (>130 lbs) per
day. Twice that for physically active person.
When [ATP] > [ADP], - G can be 11–13 Kc/Mol
Demand can change 10x in <0.001 second
Reserves are tiny. Must be able to produce large
quantities of ATP very quickly.
– ATP is more like electricity than like a battery.
Preview of ATP synthesis
Breakdown of sugars produces two moles of ATP for
each mole of sugar
– Glycolysis
Aerobic metabolism can use oxygen to create 36M
ATP/M sugar!
– Citric Acid or Tricarboxylic Acid (TCA) or Krebbs cycle.
Various compounds which can be rapidly
interconverted to ATP are used for storage
– Lipids, Glycogen, Creatinine (more like batteries)
More details when we get to catabolism...
Reductive power
Oxidation/Reduction is a necessary step in many
biosynthetic reactions
– E.g. making/breaking a double bond
Requires chemical energy
Potential carried by set of related compounds:
– Nicotinamide Adenine Dinucleotide (NAD / NADH)
• More often found in catabolism reactions
– NAD phosphate / NADPH
• More often found in anabolism reactions
– Flavin Adenine Dinucleotide (FAD / FADH)
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Coenzymes
Molecules like ATP and NADH are “helpers” to many
different kinds of enzymatic reactions
Required cofactors are called “coenzymes” and are
often “carriers” of functional groups:
– Coenzyme A carries acetyl group
– Biotin carries a carboxyl group (COO)
Vitamins are often coenzyme precursors
– Niacin (B3) is precursor to NAD
– Riboflavin (B2) to FAD
Also called “currency metabolites”
Enzymes
Enzyme activity (i.e. possible reactions) defines the
fundamental chemical steps life has at its disposal (to
turn food into babies).
In order to understand metabolism, you need to have a
sense of what enzymes can do.
Nearly all enzymes are proteins, but some RNAs and
DNAs also have enzymatic activity
Classified by the type of reaction(s) catalyzed
– Six broad groups, 3982 officially accepted classes
Oxidoreductases
Catalysts for any redox reaction
Subclassed by donor and receptor molecules
Commonly named (de-)hydrogenases and reductases
Example: Alcohol Dehydrogenase
– Oxidation of ethanol (also octanol, etc.)
– Donor is CH-OH
– Acceptor is NAD+
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Transferases
Transfer a functional group
Subclassed by what is transferred
– E.g. one carbon, nitrogen-containing, phosphate group
Example: Hexokinase. Adds
a phosphate group to a sugar
(glucose, manose, etc.)
Uses both phosphate and
energy from ATP.
Hydrolases
Special class of transferases in which water is the
acceptor.
Subclassed by the type of bond acted on
(e.g. esters, peptide bonds, etc.) and substrates
Example:
Glucose 6-phosphotase,
which undoes what
hexokinase does:
Glucose-6-phosphate + H20  Glucose + PO4
Lyases
Cleavage of a bond by other than hydrolysis or
oxidation. When acting on a single substrate, forms a
new ring or double bond
Subclassed on the bond to be broken
Commonly named “...lase” or,
when reverse reaction, “synthase”
E.g. Pyruvate decarboxylase
(double bond formed is in CO2).
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Isomerases
Catalyze changes within a molecule
(no change in composition)
Subclassed based on type of change
e.g. chirality, cis- vs. trans-, intramolecular transfers,
etc.
Example: Glucose-6-phosphate
isomerase, creates fructose6-phosphate, removing a carbon
from a 6 membered ring
Ligases
Ligases are enzymes that catalyse the joining of two
molecules
Subclassed on type of bond formed
Sometimes called “synthetase”
Example: Glutamine synthetase, adds NH4 to
Glutamate to make
Glutamine
Enzyme Kinetics
How to calculate the concentrations of reactants and
products with enzymes?
Useful (if simplified): Michaelis-Menton
– Substrate (= reactant other than enzyme) reversibly binds to
enzyme, but transition to product is irreversible:
E+S
k-1
k1
ES
kcat
E+P
– Assume [E] << [S], and, initially, [P] << [S]
– E+S
ES reaches equilibrium quickly, kcat is rate limiting
– Let the velocity of reaction V = dP/dt = kcat[ES]
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Michaelis-Menton (2)
We can define V in terms of substrate concentration
and parameters Km and Vmax
Define Km = [E][S]/[ES] = (k-1+kcat)/k1 ≅ k-1/k1
Vmax when enzyme is saturated, [E]total= [ES]
Km = [S] when V = ½ Vmax
V=
Vmax[S]
Km + [S]
V0 = Vmax[S](Km + [S])
Interpreting Enzyme Constants
Vmax and Km different for every pair of enzymes and
substrates
1/Km measures affinity of enzyme for substrate
Kcat/Km (constant part of Vmax/Km) measures efficiency
of enzyme in transforming substrate into product
Enzyme summary
Enzymes catalyze an amazing variety of reactions, but
not everything is possible
Many enzymatic reactions would be
thermodynamically unfavorable, and therefore require
coupling to -∆G (generally from ATP)
Most enzymatic reactions can run in either direction
but are used primarily in one
Kinetics can be complex, but often first order
(Michaelis-Menton) approximations are good
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