1. BMED 3510 Metabolic Systems Book Chapter 8

2. What is Metabolism? Etymology: Greek “meta · ballein” ~ to throw about, to change Metabolism is the set of life-sustaining chemical transformations within cells of living organisms. Metabolites are the intermediates and products of metabolism. The term ''metabolite'' is usually restricted to small molecules. A primary metabolite is directly involved in processes of normal growth, development, and reproduction (e.g. glucose and pyruvate). A secondary metabolite is not directly involved in those processes, but usually has an important ecological function. Examples include antibiotics and pigments. http://www.news-medical.net/; en.wikipedia.org

3. What is Metabolomics? Metabolomics is the scientific study of biochemical systems involving large numbers of metabolites at the same time. Metabolomics is the systematic study of the unique chemical fingerprints that specific cellular processes leave behind. Currently there are ≈42,000 metabolites in HMDB2, of which: 22,000 are associated with a proteins (enzyme & transporters) 5,000 have been quantified. en.wikipedia.org & www.hmdq.ca

4. Problem Assessing Metabolites The metabolome unlike the genome or the proteome is chemically very diverse. Metabolites can be water-soluble, lipid-soluble, gases, organic/inorganic, positively/negatively charged (sol)… Wide concentration range: from the molar range down to nothing. Present in several compartments (cytosol, mitochondrial, ER, ect). Half-lives of metabolites are extremely variable, with some metabolites being very short-lived inside cells

5. Assessing Metabolites Nature Protocols 6, 1241–1249 (2011)

6. Quenching in Cold Methanol Nature Protocols 6, 1241–1249 (2011)

7. Detection Http://www.bruker.com

8. Metabolic networks Http://www.infohow.org

9. Thermodynamics Prentice Hall c2002 Spontaneous reactions only occur between high and low energy metabolites. (Reactions w/ negative ΔG) Achieved by: Coupled reactions.

10. What are Enzymes? Proteins that catalyze (bio)chemical reactions

11. Why do we Have Enzymes? Higher reaction rates Most reactions don’t even occur spontaneously. Greater reaction specificity Absolute Relative (group) Stereospecificity (Stereoisomers) Milder reaction conditions Capacity for regulation Metabolites have many potential pathways of conversion or decomposition Enzymes allow substrate to be channeled into the product of highest demand

12. In the Absence of an Enzyme S P

13. In the Presence of an Enzyme S ES EP P

14. How is ∆G Lowered?

15. Catalytic Cycle of an Enzyme Substrates Products Enzyme Enzyme-substrate complex 1 Substrates enter active site; enzyme changes shape so its active site embraces the substrates (induced fit). 2 Substrates held in active site by weak interactions, such as hydrogen bonds and ionic bonds. 3 Active site (and R groups of its amino acids) can lower E A and speed up a reaction by acting as a template for substrate orientation, stressing the substrates and stabilizing the transition state, providing a favorable microenvironment, participating directly in the catalytic reaction. 4 Substrates are Converted into Products. 5 Products are Released. 6 Active site Is available for two new substrate Mole. Figure 8.17

16. Enzyme Kinetics Thermodynamics: Energy and possibility of a reaction Kinetics: Speed of a reaction Kinetics is the study of the rate at which compounds react Rate of enzymatic reaction is affected by –Enzyme –Substrate & Products –Effectors (Inhibitors & Activators) –Environmental conditions (Temperature, pH)

17. Measuring Enzyme Kinetics

18. Kinetic Properties of Enzymes Study of the effect of substrate concentration on the rate of reaction

19. Henri-Michaelis-Menten Kinetics Adian Brown (1902) Victor Henri (1903) Archibald Vivian Hill (1910) Michaelis & Menten (1913) Briggs & Aldane (1925) SE+ k1k1 k -1 SE k2k2 E+ P

20. Henri-Michaelis-Menten Kinetics Time S P ES Concentrations Assumptions: Quasi-Steady-State Assumption: Reaction mechanism Homogeneous (well stirred) k 1, k -1 >> k 2 S >> E T (E T =E+ES)

21. Henri-Michaelis-Menten Kinetics

22. [S] Reaction speed vPvP V max KMKM V max /2

23. Henri-Michaelis-Menten Kinetics K M is the apparent dissociation constant of the ES complex and measures the enzyme’s affinity for the substrate. V max = k cat E T k cat (k 2 ) is the “turnover number”: measures how many substrate molecules one enzyme molecule converts per unit of time. E T is the total enzyme concentration - K M values of enzymes differ widely. - K M provides approximation of substrate concentration in vivo. - For most enzymes, K M lies between 10 -1 and 10 -7 M. - High K M indicates weak binding. - Low K M indicates strong binding. - Most enzymes are not saturated with substrate - k cat /K M can be used as a measure of catalytic efficiency. - Diffusion limits the catalytic efficiency. Why? k cat /K M < K 1

24. Hill Kinetics [S] Reaction speed vPvP V max KMKM V max /2 Reactions catalyzed by enzymes with n subunits n n n

25. Common Enzymatic Mechanisms

26. Enzyme Inhibition Inhibitors are compounds that decrease the enzyme activity Irreversible inhibitors (inactivators) react with the enzyme - one inhibitor molecule can permanently shut off one enzyme molecule - they are often powerful toxins but also may be used as drugs Reversible inhibitors bind to, and can dissociate from the enzyme - they may be structural analogs of substrates or products - they are often used as drugs to slow down a specific enzyme Competitive inhibition: the inhibitor competes with the substrate for the active site. -Can be overcome by a sufficiently high concentration of substrate. -Inhibitor increase the Km value. Allosteric inhibition: the inhibitor binds to a different binding site.

27. Competitive Inhibition K M is modulated

28. Uncompetitive & Noncompetitive

29. Effects of Inhibitors

30. http://employees.csbsju.edu/hjakubowski/classes/ch331/transkinetics/olinhibition.html

31. http://www.as.wvu.edu/~rbrundage/lecture2b/img011.gif

32. Databases: BRENDA

33. Databases: KEGG

34. Metabolic Regulation How is metabolism regulated? Enzymatic regulation: activation or Inhibition (comp., uncomp.) Pathway regulation: - Negative feedback - Positive feedforward Isoenzymes, two or more proteins that catalyze the same reaction - Different kinetics, regulatory properties and cellular distribution - Coded by different genes or by alternative splicing Reversible covalent modification Compartmentalization Multienzyme complexes and multifunctional enzymes - Metabolite channeling - “channeling” of reactants between active sites Transcription, translation, mRNA and protein degradation

35. Metabolic Regulation How is metabolism regulated? Enzymatic regulation: activation or Inhibition (comp., uncomp.) Pathway regulation: - Negative feedback - Positive feedforward Isoenzymes, two or more proteins that catalyze the same reaction - Different kinetics, regulatory properties and cellular distribution - Coded by different genes or by alternative splicing Reversible covalent modification Compartmentalization Multienzyme complexes and multifunctional enzymes - Metabolite channeling - “channeling” of reactants between active sites Transcription, translation, mRNA and protein degradation

36. Pathway Regulation Product of a pathway controls the rate of synthesis by inhibiting an early step, usually the first “committed” step (unique to the pathway). Metabolite early in the pathway activates an enzyme further down the pathway Negative feedback Positive feedforward

37. Glycolysis in L. lactis A.R. Neves et al. FEMS Microbiol. Rev. 29 (2005) 531–554

38. Metabolic Regulation How is metabolism regulated? Enzymatic regulation: activation or Inhibition (comp., uncomp.) Pathway regulation: - Negative feedback - Positive feedforward Isoenzymes, two or more proteins that catalyze the same reaction - Different kinetics, regulatory properties and cellular distribution - Coded by different genes or by alternative splicing Reversible covalent modification Compartmentalization Multienzyme complexes and multifunctional enzymes - Metabolite channeling - “channeling” of reactants between active sites Transcription, translation, mRNA and protein degradation

39. Amino Acid Biosynthesis http://www.uky.edu/~dhild/biochem/24/lect24.html

40. Metabolic Regulation How is metabolism regulated? Enzymatic regulation: activation or Inhibition (comp., uncomp.) Pathway regulation: - Negative feedback - Positive feedforward Isoenzymes, two or more proteins that catalyze the same reaction - Different kinetics, regulatory properties and cellular distribution - Coded by different genes or by alternative splicing Reversible covalent modification Compartmentalization Multienzyme complexes and multifunctional enzymes - Metabolite channeling - “channeling” of reactants between active sites Transcription, translation, mRNA and protein degradation

41. Reversible Covalent Modification

43. Metabolic Regulation How is metabolism regulated? Enzymatic regulation: activation or Inhibition (comp., uncomp.) Pathway regulation: - Negative feedback - Positive feedforward Isoenzymes, two or more proteins that catalyze the same reaction - Different kinetics, regulatory properties and cellular distribution - Coded by different genes or by alternative splicing Reversible covalent modification Compartmentalization Multienzyme complexes and multifunctional enzymes - Metabolite channeling - “channeling” of reactants between active sites Transcription, translation, mRNA and protein degradation

44. Insulin-Induced Glucose Uptake

45. Metabolic Regulation How is metabolism regulated? Enzymatic regulation: activation or Inhibition (comp., uncomp.) Pathway regulation: - Negative feedback - Positive feedforward Isoenzymes, two or more proteins that catalyze the same reaction - Different kinetics, regulatory properties and cellular distribution - Coded by different genes or by alternative splicing Reversible covalent modification Compartmentalization Multienzyme complexes and multifunctional enzymes - Metabolite channeling - “channeling” of reactants between active sites Transcription, translation, mRNA and protein degradation

46. Multifunctional Enzymes

47. Metabolic Regulation How is metabolism regulated? Enzymatic regulation: activation or Inhibition (comp., uncomp.) Pathway regulation: - Negative feedback - Positive feedforward Isoenzymes, two or more proteins that catalyze the same reaction - Different kinetics, regulatory properties and cellular distribution - Coded by different genes or by alternative splicing Reversible covalent modification Compartmentalization Multienzyme complexes and multifunctional enzymes - Metabolite channeling - “channeling” of reactants between active sites Transcription, translation, mRNA and protein degradation

48. Summary Advantages of having enzyme catalyzed reactions How do enzymes operate? Michaelis-Menten rate law (approximation) Effect of different types of inhibition Metabolic regulation many modes, operating at different time scales