Temperature

1. Temperature (I will focus on Adaptation of Enzymes)

2. Outline (1) Physical and Physiogical Effects of Temperature (Q10) (2) Evolution of Enzyme Function

3. Physical Forces in the Environment • Physical factors in the Environment (temperature, salinity, light, oxygen, etc) impose selective forces • to which the organism must respond

4. Physical Properties of Water and Air PropertyWaterAir HumidityHighLow DensityHigh (800x) Low ViscosityHigh (50x) Low Heat Capacity High (3000x) Low O2 SolubilityLowHigh (30x) O2 DiffusivityLowHigh (8000x) Light ExtinctionHighLow

5. The range of temperature in water is less than…

6. The range of temperature on land

7. Range of temperatures on earth far exceed the range compatible with animal life

8. Rate of Reaction or Biological Process Rate Enhancing Effects Destructive Effects Optimal Temperature Temperature

9. Q10roughly describes effects of temperature on physiology Q10 = the effect of temperature on physiology at one temperature versus another 10°C different

10. Q10 roughly describes effects of temperature on physiology Higher Temperature Increase in activity of molecules Faster chemical reactions

11. Q10 roughly describes effects of temperature on physiology Physiological processes could include metabolic rate, ingestion rate, digestion rate, etc…

12. Q10 = 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

13. 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

14. But… • What other environmental variables might vary with temperature?

15. 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

16. 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!!!

17. EXAMPLE: The coupling of temperature with fluid viscosity can greatly impact physiological processes at small scales (low Reynolds Numbers)

18. So how much of Q10 is due to the effect of temperature alone, versus the effects of a covariable, such as viscosity?

20. Separated Effects of Temperature and Viscosity by adding Dextran… dextran changes viscosity without changing temperature

21. Add Dextran to artificially raise viscosity Independent of temperature Relationship between viscosity and temperature

22. Mean number of particles ingested over 10 minute trials Viscosity manipulated by adding dextran

23. Temperature is 22°C, but viscosity is equivalent to that of 12°C (by adding Dextran)

24. About 60% of difference in performance is due to effects of Viscosity alone!!!!

25. 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)

26. Outline (1) Physical and Physiogical Effects of Temperature (Q10) (2) Evolution of Enzyme Function

27. Enzymes

28. 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) Terms

29. 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

30. 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

31. 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

32. k1 k2 E + S E  S E + P 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

33. Michaelis-Menten Equation Vmax [S] Km + [S] Velocity (rate of reaction) = Km = substrate affinity, where Vmax/2 Also called “Michaelis-Menten constant” [S] = substrate concentration Vmax = maximum velocity

34. Michaelis-Menten Equation Vmax [S] Km + [S] 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.

35. k1 kcat E + S E  S E + P k-1 Enzyme Reaction • 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

36. 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 Catalytic Efficiency Vmax [E]t kcat =

37. Adaptive Response of Enzymes

38. Evolutionary Shifts in Reaction Norms

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

40. Fundulus heteroclitus 15°C difference in Mean Temperature along Atlantic Coast

41. LDH

43. 1° latitude change = 1°C change in mean water temperature The two alleles of LDH have a latitudinal distribution Place and Powers, PNAS 1979

44. 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

45. 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)

46. kcat/Km is larger for the b allele at low temperatures aagenotype ab LHD-B b anda 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 bb Place and Powers, 1979

47. aa genotype ab LHD-B b anda alleles have different catalytic efficiencies (kcat/Km) at different temperatures Or they show what is called, “genotype by environment interaction” bb Place and Powers, 1979

48. 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 The structural differences between the alleles seem to affect function Weakness of this study? Fish DiMichele et al. 1991 Science

49. 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)

50. Schulte et al. 2000 This difference in expression is due to the presence of a regulatory element (an enhancer) Enhancer present 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)