Carol Eunmi LeeUniversity of Wisconsin

Evolution 410Copyright ©2020; do not upload without permission

Protein Evolution

So… different types of mutations could include:

• STRUCTURAL: Affects the Property of the gene product (changes to the allele itself)– A mutation could occur within the coding sequence of a gene,

and change the amino acid composition of a protein (structural)

• REGULATORY: Typically affects the Amount of the gene product– A mutation could occur within a regulatory element, like a

promoter or enhancer near the gene (cis-regulatory)– A mutation could occur within a regulatory element, like a

transcription factor that is encoded far away from the gene (trans-regulatory)

Loss of function of extra gene copy

New function of extra gene copy

Partition of function between the gene copies

Fate of Duplicated Genes

• Paralogs: duplicated genes in a genome, followed by differentiation

• Orthologs: homologous genes in different populations or species

Outline(1) Types of Mutations that could affect

functionStructural ChangesRegulatory Changes

(2) Case study of structural changes (protein evolution) à and how it affects temperature adaptation of the enzyme LDH

STRUCTURAL evolutionary changes

• Mutations in DNA or mRNA that result in changes in the amino acid composition of a protein

• Some amino acid changes alter the activity and/or function of proteins (and enzymes)

Diagram of eukaryotic gene Each gene is composed of

regulatory and coding region

Eukaryotic gene (DNA)

Futuyma D.J. (2009)

“The Central Dogma” of Molecular Biology

Francis Crick (1958)

Codon Bias

§ In the case of amino acids

§ Mutations in Position 1, 2 lead to Amino Acid change

§ Mutations in Position 3 often don’t matter

A Classic Example: temperature adaptation in the killifish Fundulus heteroclitus

LDH

LDH is a glycolytic enzyme which catalyzes the reaction between Pyruvate and Lactate

Protein function

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

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

changes, receptors)

Fundulus heteroclitus

Populations in Maine and Georgia have different proportions of alleles (isozymes) at LDH-B

• In cold temperature generally activity of an enzyme generally slows down

• So, in cold temperature, enzymes generally compensate, to make up for the slower function.

• How?

• In hot temperature, enzymes have higher activity, but can denature more readily.

Temperature Adaptation and Tradeoffs in Enzyme Function

Example: Enzyme Functional Evolution

• Enzymes are proteins that lower the activation energy (Ea) of a chemical reaction (“catalyzes the reaction”)

• Different isozymes (enzymes encoded by different alleles) 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

Example: Enzyme Functional Evolution

Important properties of the enzyme that could evolve:

• KM

• Vmax

• kcat = Vmax[E]

• Catalytic Efficiency

Example: Enzyme Functional Evolution

Important properties of an enzyme that could evolve:

• KM : an inverse measure of the substrate's affinity for the enzyme (small KM indicates high affinity)

• Vmax: maximum reaction velocity

• kcat = VmaxConcentration of Enzyme sites

• Catalytic Efficiency = kcat / KM

E + S E + PE • Sk1 k2

k-1

Enzyme Reaction

E = enzymeS = substrateP = product

where

E • S = enzyme-substrate complexk1 , k-1 , k2 = enzyme reaction ratesk2 is also called kcat, the catalytic constant

Michaelis-Menten Equation

Velocity (rate of reaction, dP/dt) =

KM = substrate affinity; the substrate concentration 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]

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 à greater rate of reaction

• The ratio of kcat / KM is a measure of the enzyme’s catalytic efficiency

Vmax[E]t

kcat =

E + S E + PE • Sk1 kcat

k-1

Enzyme Reaction

E = enzymeS = substrateP = product

where

E • S = enzyme-substrate complexk1 , k-1 , k2 = enzyme reaction ratesk2 is also called kcat, the catalytic constant

KM

Video with good explanation of KM

https://www.youtube.com/watch?v=rCVRC-AQ54M

How the Michaelis-Menten equation is derived:https://www.youtube.com/watch?v=FXWZr3mscUo

E + S E + PE • Sk1 kcat

k-1

Enzyme Reaction

• There could be evolutionary differences in KM or kcatin different habitats

• And KM and kcat among populations or species could evolve

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

KM

• In cold temperature generally activity of an enzyme generally slows down

• So, in cold temperature, enzymes generally compensate, to make up for the slower function.

• How? –increase in kcatà increase in rate of reaction• but, KM will increase (lower structure integrity)

• In hot temperature, enzymes have higher activity, but can denature more readily. —want to increase stability (lower KM, lower kcat)

Temperature Adaptation and Enzyme Function

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

Place and Powers, PNAS 1979

Place and Powers, PNAS 1979

Different alleles (isozymes) predominate in North vs South

North: LDH-B b allele (cold-adapted)

South: LDH-B a allele (warm-adapted)

The two alleles (proteins) differ at 2 amino acids

Place and Powers, 1979

b allele homozygotea allele homozygote

Catalytic efficiency (kcat/KM) is higher for the b allele at low temperature, and higher for the a allele at higher temperature

kcat/KM

Place and Powers, 1979

• The two allele products (the enzymes) show genetic differences in catalytic efficiency (adaptive differences) across temperatures

• They also show Genotype xEnvironment interactions and evolutionary tradeoffs in function across different temperatures, with the bb homozygote doing better in the cold, and the aahomozygote doing better at higher temperature

Catalytic efficiency (kcat/KM) is higher for the b allele at low temperature, and higher for the a allele at higher temperature

kcat/KM

There are many possible limitations (costs or constraints) preventing complete adaptation to an environment due to evolutionary tradeoffs

For enzyme function, there is often a tradeoff between functional capacity (indicated by Vmax) and enzyme stability(KM is one measure)

Evolutionary Tradeoff in enzyme function at cold vs high temperatures

Tradeoff between flexible vs stable enzyme structure

• Cold Temperature:• Flexible, can have higher activity to compensate for

cold temperature (higher kcat)• But hard to maintain structural integrity at high

temperature (higher KM)

• Warm Temperature: • Stable, to maintain structural integrity at high

temperature (lower KM)• But, lower enzyme activity (ok, because temperature

is high) (lower kcat under common garden conditions)

In damsel fish LDH, a tradeoff between functional capacity and enzyme stability has been found

More cold-adapted enzymes are labile (flexible, higher kcat) and less stable at higher temperatures

More warm-adapted enzymes have been found to be more stable, but less flexible

In damsel fish LDH, a tradeoff between functional capacity and enzyme stability has been found

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 bJ-a1G 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

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

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

Threonine is more hydrophilic and thought to make the loop more flexible (higher Vmax)

The Alanine amino acid substitution causes KM and kcatto be reduced in the tropical orthologs

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

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

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

KM and kcat are higher in the cold-adapted ortholog

Chemical reactions slow down in colder temperature environments

àNeed a more flexible enzyme

With a high rate of reaction to compensate for the slowed down rates of reaction in the coldIn cold habitats, you need to compensate for lower rates of reaction activity by making the enzyme more flexible à high kcatsacrifice substrate affinity (high KM)or, fast &sloppy enzymes;the cold temperature will keep the floppy enzyme more stable

Higher enzyme reaction rate in cold water fish

Lower enzyme stability in cold water fish

KM

kcat

KM and kcat are lower in the warm-adapted ortholog

Chemical reactions are faster in warm temperature environments

Need a more rigid enzyme, so it does not denature in the heat

Such an enzyme will have a lower rate of reaction

Warmer (black square, triangle): Less flexible (low kcat), but higher binding ability (low KM)

Lower enzyme reaction rate in warm water fish

Higher enzyme stability (low KM) in warm water fish

KM

kcat

Tradeoffs between Enzyme lability and stability:

Colder (white circles): more flexible (high kcat), but loss of binding ability (high KM)

Warmer (black squares, triangles): Less flexible (low kcat), but higher binding ability (low KM)

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

SummaryRates of enzyme reaction speed up in higher temperatures and slow down in colder temperatures

In low temperatures need greater enzyme lability to compensate for slower reaction rates in the coldàselection will favor enzymes with higher KM and

higher kcat (higher Vmax)

In high temperatures need greater enzyme stability (to prevent denaturation at high temperatures) à selection will favor enzymes with lower KM and lower kcat

Patterns of Molecular Evolution• What are mutations? How would structural vs regulatory

mutations affect function?

• What are the possible targets of selection for LDH in response to temperature?

• How does temperature affect Enzyme Kinetics?

• What changes in enzyme function might enhance a response to an environmental variable (such as temperature)? (Vmax, KM, kcat, kcat/KM)

• Why are there tradeoffs between enzyme reaction rates (functional capacity) and stability?

• Why are there tradeoffs between cold and warm adaptation in enzyme function?

• How might organisms evolve in response to global warming? What about global cooling?

1. When comparing DNA sequences that encode a protein between two species, the number of substitutions at nonsynonymous was found to be much higher than those at synonymous sites. This result suggests evidence for:

(a) Non-adaptive evolution(b) Adaptive evolution(c) Negative selection(d) Evolutionary constraint(e) Preferential fixation of synonymous sites

2. Which of the following is FALSE regarding the functional differences among the enzymes above?

(a) The different genotypes appear to show tradeoffs between functioning well (higher catalytic efficiency) at cold vs warmer temperatures

(b) The performance of the three genotypes shows no evidence for heterozygote advantage

(c) Adaptation to temperature in these enzymes is likely due to differences in amino acid composition between the proteins encoded by the a versus balleles

(d) kcat/Km is higher for the aa genotype than for the bb genotype at warmer environments

(e) Differences in allelic function above reflect structural evolutionary changes and prove that regulatory changes have not occurred

The graph shows the catalytic efficiency (kcat/Km) for three genotypes of the LDH-B enzyme across temperatures for populations of the fish Fundulus heteroclitus.

3. A fragment of DNA from an LDH-B allele shows higher number of nonsynonymous relative to synonymous substitutions than expected. When this fragment is injected into a fish, it shows elevated pyruvate metabolism relative to the equivalent fragment from another allele. (a) Evolutionary Adaptation (structural change)(b) Evolutionary Adaptation (regulatory change)(c) Linkage(d) Physical/functional constraint(e) Insufficient information to determine

4. Which enzyme would best compensate for the challenges of living in a warmer environment?

(a) A rigid enzyme

(b) An enzyme with a comparatively higher Vmax

(c) An enzyme with a high Km

(d) A more labile enzyme with a low Vmax

(e) An enzyme with a lower kcat/Km

• 1B• 2E• 3A• 4A