Outline - University of Wisconsin–Madison

Carol Eunmi Lee 1/25/11

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Outline

(1) Physical and Physiogical Effects of Temperature (Q10)

(2) Evolution of Enzyme Function

Physical Forces in the Environment

  Physical factors in the Environment (temperature, salinity, light, oxygen, etc) impose selective forces

  to which the organism must respond

The range of temperature in water is less than…

The range of temperature on land


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Optimal Temperature

Rate of Reaction

or Biological Process

Temperature

Rate Enhancing Effects

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


Higher Temperature

Increase in activity of molecules

Faster chemical reactions


Physiological processes could include metabolic rate, ingestion rate, digestion rate, etc…

= rate at T+10°C = rate at T rateT 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   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

273 283 2.00 373 383 1.45 473 483 1.26

But…

  What other environmental variables might vary with temperature?

Important Point when thinking about environmental variables:   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:   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!!!

So how much of Q10 is due to the effect of temperature alone, versus the effects of a covariable,

such as viscosity?


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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

Viscosity manipulated by adding dextran

Mean number of particles ingested over 10 minute trials

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!!!!


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Lesson   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

Terms   Paralogs: genes related by duplication within a genome.

Following duplication, they often experience subfunctionalization, neofunctionalization, or loss of function

  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.

  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.

  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

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

k = A e-Ea/RT


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•  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

E + S E + P E • S k1 k2

k-1

Enzyme Reaction

E = enzyme S = substrate P = product

where

E • S = 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) =

Km = substrate affinity, where Vmax/2

Also called “Michaelis-Menten constant”

[S] = substrate concentration

Vmax = maximum velocity

Vmax [S]

Km + [S]

Michaelis-Menten Equation

Velocity (rate of reaction) =

•  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.

Vmax [S]

Km + [S]

E + S E + P E • S k1 kcat

k-1

Enzyme Reaction

•  There could be evolutionary differences in Km

•  And kcat among species could evolve

•  kcat depends on the ΔG (activation free energy) of the chemical reaction

Catalytic Efficiency   Catalytic constant, kcat :

  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

Vmax

[E]t kcat =


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Adaptive Response of Enzymes

Evolutionary Shifts in

Reaction Norms

Km and kcat of A4LDH orthologs vary among species adapted to different temperatures

LDH


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Place and Powers, PNAS 1979

The two alleles of LDH have a latitudinal distribution

1° latitude change = 1°C change in mean water temperature

Enzyme function could evolve via changes in

STRUCTURE   Amino acid composition (AA substitutions)   Secondary, Tertiary, Quaternary structure

REGULATORY   Protein expression (transcription, translation, etc)   Protein activity (allosteric control, conformational changes,

receptors)

  Review Lectures on adaptation

Adaptation of LDH-B to temperature

  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)

Place and Powers, 1979

aa genotype ab

bb

kcat/Km is larger for the b allele at low temperatures

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”

Fish

Significantly different rates of glucose uptake depending on whether the eggs were injected with the “a” versus “b” allele

The structural differences between the alleles seem to affect function

Weakness of this study?

DiMichele et al. 1991 Science

The Allozymes show differences in Function


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But, differences in gene (protein) expression of the two alleles might also be important

  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)

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)

Schulte et al. 2000

control

Enhancer present

This difference in expression is due to the presence of a regulatory element (an enhancer)

Functional Tradeoffs

Functional capacity vs Enzyme stability

Cold vs Warm adapted enzymes

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?

Fish

kcat values are higher in species adapted to colder temperatures

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


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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 βJ-α1G 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

Threonine is more hydrophilic and thought to make the loop more flexible

Johns and Somero 2004

Chromis caudilis (tropical, warmer)

Chromis punctipinnis (temperate, colder)

Chromis xanthochira (tropical, warmer)

Higher reaction rate in colder fish

Lower stability in colder fish

Km

kcat

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)

Johns and Somero 2004

Chromis caudilis (tropical)

Chromis punctipinnis (temperate)

Chromis xanthochira (tropical)

When cold, you need to compensate for lower 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

Higher reaction rate in colder fish

Lower stability in colder fish

Km

kcat

Extra credit   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


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  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

  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 following is a graph showing functional responses for four different enzymes (a, b, c, d).

14. Which of the enzymes has lowest substrate affinity? 15. Which of the enzymes has greatest catalytic efficiency (kcat / Km)?

Example of an exam question that tests

whether the student understands the

concepts

Email your extra credit assignment to:

[email protected] by Saturday Feb 5, 5 pm

Put: “extra credit” in the subject heading Make sure your name is clearly stated in the email

(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)?

Study Questions

(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?

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Outline - University of Wisconsin–Madison