Temperature
(I will focus on Adaptation of Enzymes)
Outline
(1) Physical and Physiogical Effects of
Temperature (Q10)
(2) Evolution of Enzyme Function
Physical Forces in the Environment
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Physical factors in the
Environment (temperature,
salinity, light, oxygen, etc) impose
selective forces
to which the organism must
respond
Physical Properties of Water and Air
Property
Humidity
Water
High
Air
Low
Density
High (800x)
Low
Viscosity
High (50x)
Low
Heat Capacity
High (3000x)
Low
O2 Solubility
Low
High (30x)
O2 Diffusivity
Low
High (8000x)
Light Extinction
High
Low
The range of temperature in water is less than…
The range of temperature on land
Range of temperatures on earth far exceed
the range compatible with animal life
Rate of
Reaction
or
Biological
Process
Rate Enhancing
Effects
Optimal
Temperature
Temperature
Destructive
Effects
Q10 roughly describes
effects of temperature
on physiology
Q10 = the effect of temperature
on physiology at one
temperature versus another
10°C different
Q10 roughly describes effects of temperature on
physiology
Higher Temperature
Increase in activity of
molecules
Faster chemical
reactions
Q10 roughly describes effects of temperature on
physiology
Physiological
processes could
include metabolic rate,
ingestion rate,
digestion rate, etc…
Q10
=
rate at T+10°C
rateT
=
rate at T
rateT-10°C
= ratio of the rate of a reaction at one temperature divided by
the rate of the same reaction at a temperature 10 C° less.
Larger the Q10 = greater effect of temperature on rate of
reaction.
Q10 = 1 implies no effect of temperature on the rate of
reaction.
Typical Q10 values = 2 ~ 4
Q10
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Q10 is only a very rough indication of the effect of
temperature on physiological activity
At greater temps, difference might be the same but
ratio might decrease
– Temperature in Kelvins
Ratio
T1/K
T2/K
k2/k1
273
373
473
283
383
483
2.00
1.45
1.26
But…
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What other environmental variables
might vary with temperature?
Important Point when thinking
about environmental variables:
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Environmental variables can covary or interact with other
variables
For example, temperature covaries with a lot of other
variables
Such as Viscosity, Oxygen concentration, pH, Solubility of a
chemical, etc.
These other variables might also affect physiological
processes
If you aren’t careful, effects of these other variables might be
confused with effects of temperature
Important Point when thinking
about environmental variables:
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With increase water temperature, oxygen concentration
declines (Charles’ Law)
With increasing temperature, CO2 concentration decreases,
and blood pH increases
With increasing temperature, viscosity declines
When you think you are testing for the effects of
Temperature, you might actually be measuring the
effects of something else!!!
EXAMPLE: The coupling of
temperature with fluid viscosity
can greatly impact physiological
processes at small scales (low
Reynolds Numbers)
So how much of Q10 is due to the effect
of temperature alone, versus the
effects of a covariable,
such as viscosity?
Separated Effects of Temperature and Viscosity
by adding Dextran… dextran changes viscosity without
changing temperature
Add Dextran to artificially raise viscosity
Independent of temperature
Relationship
between
viscosity and
temperature
Mean number of particles ingested over 10 minute trials
Viscosity manipulated by adding dextran
Temperature is 22°C, but viscosity is equivalent to
that of 12°C (by adding Dextran)
About 60% of difference in performance
is due to effects of Viscosity alone!!!!
Lesson
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Many Physical Variables covary
When you are testing the effect of a variable (such
as temperature) keep in mind that you could also be
changing other variables
(such as O2 conc., viscosity, pH, etc)
Examine interaction term among variables in an
analysis of variance (ANOVA)
Outline
(1) Physical and Physiogical Effects of
Temperature (Q10)
(2) Evolution of Enzyme Function
Enzymes
Terms
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Paralogs: genes related by duplication within a genome.
Following duplication, they often experience subfunctionalization,
neofunctionalization, or loss of function
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Orthologs: genes in different species that evolved from a
common ancestral gene by speciation. Often, orthologs retain the
same function during the course of evolution.
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Isozymes: different forms of the same enzyme, usually resulting
from gene duplications (paralogs); they often differ in amino acid
sequence but catalyze the same chemical reaction. These enzymes
usually display different kinetic parameters (i.e. different Km values),
or different regulatory properties.
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Allozymes: enzyme products of different alleles of the same gene
(allelic enzymes at a locus)
Sample exam question
I have two closely related detoxification
enzymes, that are nearby on the same
chromosome. One breaks down cocaine and
the other breaks down caffeine. These
proteins are:
(A) paralogs
(B) orthologs
(C) isozymes
(D) allozymes
The Arrhenius Equation
k = A e-Ea/RT
The rate constant k of a chemical reaction depends on
temperature T (in Kelvins) and activation energy Ea:
A = pre-exponential factor
R = gas constant
Ea = activation energy, minimum amount of energy required
to transform reactants into products
• Enzymes lower the activation energy (Ea) of a
chemical reaction (“catalyzes the reaction”)
• Different isozymes with different properties would
lower the activation energy to differing degrees
• That is, enzymes with different Km or kcat will lower Ea
to differing degrees
Enzyme Reaction
k1
E + S
ES
k2
E + P
k-1
where
E
S
P
ES
= enzyme
= substrate
= product
= enzyme-substrate complex
k1 , k-1 , k2 = enzyme reaction rates
k2 is also called kcat, the catalytic constant
Michaelis-Menten Equation
Velocity (rate of reaction) =
Vmax [S]
Km + [S]
Km = substrate affinity, where Vmax/2
Also called “Michaelis-Menten constant”
[S] = substrate concentration
Vmax = maximum velocity
Michaelis-Menten Equation
Velocity (rate of reaction) =
Vmax [S]
Km + [S]
• Small Km: enzyme requires only a small amount of
substrate to become saturated. Hence, the maximum
velocity is reached at relatively low substrate
concentrations. (greater substrate binding specificity)
• Large Km: Need high substrate concentrations to
achieve maximum reaction velocity.
Enzyme Reaction
k1
E + S
ES
kcat
E + P
k-1
• There could be evolutionary differences in Km
• And kcat among species could evolve
• kcat depends on the G (activation free energy) of the
chemical reaction
Catalytic Efficiency
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Catalytic constant, kcat :
kcat =
Vmax
[E]t
kcat = turnover number = the rate at which substrate is
converted to product, normalized per active enzyme site; Et is
the concentration of enzyme sites you've added to the assay
High kcat  greater rate of reaction
The ratio of kcat / Km is a measure of the enzyme’s catalytic
efficiency
Adaptive Response of Enzymes
Evolutionary
Shifts in
Reaction Norms
Km and kcat of A4LDH
orthologs vary among
species adapted to
different temperatures
Fundulus heteroclitus
15°C difference in Mean Temperature
along Atlantic Coast
LDH
1° latitude change
= 1°C change in mean
water temperature
The two alleles of
LDH have a latitudinal
distribution
Place and Powers, PNAS 1979
Enzyme function could
evolve via changes in
STRUCTURE

Amino acid composition (AA substitutions)
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Secondary, Tertiary, Quaternary structure
REGULATORY
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Protein expression (transcription, translation, etc)
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Protein activity (allosteric control, conformational changes,
receptors)
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Review Lectures on adaptation
Adaptation of LDH-B to temperature
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There are structural differences in the
enzymes (in amino acid composition between
a vs. b alleles, allozymes)
Differences in amino acid composition could
result in functional differences in the enzyme
Enzyme kinetics of the allelic products (aa,
ab, bb) differ in this case (that is, specific
activity of the enzymes differ in different
environments)
kcat/Km is larger for the b
allele at low temperatures
aa genotype
ab
bb
LHD-B b and a
alleles have different
catalytic efficiencies
(kcat/Km) at different
temperatures
Or they show what is
called, “genotype by
environment
interaction”, i.e.
different genotypes do
different things in
different environments
Place and Powers, 1979
aa genotype
ab
bb
LHD-B b and a
alleles have different
catalytic efficiencies
(kcat/Km) at different
temperatures
Or they show what is
called, “genotype by
environment
interaction”
Place and Powers, 1979
The Allozymes show differences in Function
Significantly different
rates of glucose
uptake depending on
whether the eggs
were injected with
the “a” versus “b”
allele
Fish
DiMichele et al. 1991 Science
The structural
differences between
the alleles seem to
affect function
Weakness of this
study?
But, differences in gene (protein)
expression of the two alleles might
also be important
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Differences in gene expression could be caused by
differences in the promoter, enhancer or some other
regulatory element
NOT by differences in the nucleotide composition of
the gene itself (by the amino acid composition of the
protein)
Enhanced expression leads to greater number of
copies of the gene being transcribed (and then
translated into protein)
Schulte et al. 2000
This difference in
expression is due to
Enhancer present
the presence of a
regulatory element
(an enhancer)
control
Figure: Transgenic Fish
Regulatory sequence (an enhancer)
injected into Northern or Southern Fish
The regulatory sequence is contained
within the 500, but not 400 base pair
sequence
The Northern regulatory sequence
enhances LDH activity when injected
into both Northern and Southern fish
(experiment performed at 20°C)
Functional Tradeoffs
Functional capacity vs Enzyme stability
Cold vs Warm adapted enzymes
kcat values are
higher in species
adapted to colder
temperatures
Fish
For many species (mammals, birds, reptiles, fish), orthologs
of A4LDH of cold-adapted species are more effective at
lowering activation energy (Ea values) than those of warm
adapted species (Fields and Somero, 1998)
So then, why not have these more effective cold-adapted
enzymes in all environments?
There are many possible limitations (costs
or constraints) preventing complete
adaptation to an environment (see paper
by Somero)
One possibility is the tradeoff
between functional capacity and
enzyme stability
Tradeoff between
functional capacity
and enzyme stability
More cold-adapted enzymes
are labile (flexible, higher
kcat) and less stable at higher
temperatures
If too unstable, lose
geometry for ligand
recognition and binding
(higher Km)
Protein could become
inactivated
Tradeoff between
functional capacity
and enzyme stability
Dark areas experience
conformational changes
during ligand binding, such
that amino acid changes
here could affect enzyme
function (kcat or Km)
This Thr -> Ala amino acid
substitution corresponds to
temperate -> tropical shift
A4LDH
This Thr -> Ala amino acid
substitution, at position 219
in the J-1G loop of
A4LDH, corresponds to
temperate -> tropical shift in
Damselfish
Threonine is more
hydrophilic and thought
to make the loop more
flexible (higher Km, kcat)
Threonine -> Alanine amino
acid substitution at a catalytic
loop corresponds to temperate
-> tropical shift in Damselfish
Km and kcat are higher in the
temperate (colder) ortholog
The Alanine amino acid
substitution causes Km and
kcat to be reduced in the
tropical orthologs
Km
Lower stability in colder fish
Chromis punctipinnis
(temperate, colder)
Chromis caudilis
(tropical, warmer)
kcat
Higher reaction rate in colder fish
Threonine is more hydrophilic
and thought to make the loop
more flexible
Johns and Somero 2004
Chromis xanthochira
(tropical, warmer)
Km
Tradeoffs:
Colder (white circles): more
flexible (high kcat), but loss of
binding ability (high Km)
Warmer (black square,
triangle): Less flexible (low
kcat), but higher binding ability
(low Km)
Lower stability in colder fish
Chromis punctipinnis
(temperate)
When cold, you need to
Chromis caudilis
compensate for lower
(tropical)
rates of reaction activity by
making the enzyme more
flexible  high kcat sacrifice
Km (high Km)
or, fast &sloppy enzymes;
the cold will keep enzyme
more stable
Chromis xanthochira
(tropical)
kcat
Higher reaction rate in colder fish
Johns and Somero 2004
Summary
(1) Physiological responses tend to increase with
temperature, until they are limited by destructive effects
of high temperature
(2) It is important to remember that many other physical
variables covary with temperature such that experiments
can be confounded by multiple variables
(3) Enzyme activity is affected by temperature, and
enzymes can evolve function in response to
temperature, such as altering amino acid composition,
conformation, or gene and protein expression
(4) There are tradeoffs between enzyme lability and
stability
Extra credit
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Extra credit points will be given for sample exam
questions from the course material
1-3 pts will be given for each question, for up to 6
points
For 2 questions
The questions must test thought and understanding,
rather than simple regurgitation

Example of a question involving regurgitation:
Which of the following parameters indicates the
substrate affinity of an enzyme?
(a) kcat (b) Km (c) Ea (d) Vmax
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This is a good question but does not require an
understanding of what the terms mean
This question would receive 0 points
Also, 0 points for plagiarized questions, or those
identical to your classmates.
The foll owing is a graph showing functional responses for four different enzymes (a, b, c, d).
Example of an exam
question that tests
whether the student
understands the
concepts
14. Which of the enzymes has lowest substrate affi nity?
15. Which of the enzymes has greatest catalytic efficiency (kcat / K m)?
Email your extra credit assignment to:
carollee@wisc.edu by Saturday Feb 5, 5 pm
Put: “extra credit” in the subject heading
Make sure your name is clearly stated in the email
Study Questions
(1) Discuss Adaptations to high and low temperature at
multiple hierarchical levels (amino acid substitution, gene
duplications, etc).
(2) What is Q10? If oxygen consumption of an animal is 10
mol/s at 15°C, and 20 mol/s at 25°C, what is the Q10
of this physiological activity? What does this Q10 value
mean?
(3) What other environmental variables interact with
temperature, and how might a confounding physical
variable affect the measurement of temperature effects on
physiology (such as Q10)?
(4) What are the possible targets of selection for LDH in
response to temperature?
(5) How does temperature affect Enzyme Kinetics?
(6) What changes in enzyme function might enhance a
response to an environmental variable (such as
temperature)? (Vmax, Km, Kcat, Kcat/Km, etc??)
(7) Why are there tradeoffs between enzyme function and
stability?
(8) Why are there tradeoffs between cold and warm adaptation
in enzyme function?
(9) Would global warming have the same or different effect on
terrestrial versus aquatic organisms? Why? What about
global cooling?