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Protein Structure, Function and The Enzymes of Glycolysis

Protein Structure, Function and The Enzymes of Glycolysis. triosephosphate isomerase. How Proteins Work. Proteins recognize and bind to other molecules. The bound molecule is called a ligand . The region of a protein that associates with substrates and products is called the active site.

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Protein Structure, Function and The Enzymes of Glycolysis

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  1. Protein Structure, Function and The Enzymes of Glycolysis triosephosphate isomerase

  2. How Proteins Work Proteins recognize and bind to other molecules. The bound molecule is called a ligand. The region of a protein that associates with substrates and products is called the active site. The region of a protein that associates with activator or inhibitor molecules is called an allosteric site.

  3. hexokinase catalytic domain This model was created in Chemscape Chime from the 2YHX pdb file by C.M.ANDERSON,R.E.STENKAMP,T.A.STEITZ. Red indicates a helix. Yellow indicates b sheets. OTG (a glucose analog shown in white) is bound at the active site.

  4. Proteins fold in such a way that they create specific sites that are the right size, shape, and polarity for their ligands.

  5. Triosephosphate isomerase Substrate = Dihydroxy acetone phosphate

  6. Ligand binding is highly selective

  7. The binding site is created by non covalent interactions between the ligand and specific amino acid side chains

  8. Michaelis-Menton hyperbolic kinetics Vmax Km The hyperbolic curve is defined by two parameters: Vmax and Km

  9. Maximum velocity or Vmaxis the maximum velocity of the reaction when the enzyme is saturated with substrate. Turnover rate:the number of substrate molecules converted to product per second. catalasehas a turnover rate of 93,000. DNA polymerasehas a turnover rate of 15.

  10. Kmis the substrate concentration at which the reaction velocity is equal to one half the maximal velocity (Vmax). Values for (Km) are in the range of 10-1 to 10-7 M.

  11. [products] [reactants] D G= D G0 + RT ln Many reactions are shared. For these, DG0 is usually either slightly positive or slightly negative. Thus, the direction of the reaction is dependent on the [reactant] and [product]. For example: G6P F6P DG0 = + 1.7 kJ/mole; DG = -2.5 kJ/mole

  12. glucose glucose glycolysis gluconeogenesis pyruvate pyruvate Many steps are shared. But, parallel pathways of catabolism and anabolism must differ in at least one step.

  13. A The enzymes that catalyze the reactions that are different are targets for allosteric regulation. B C D E F Allosteric means “different site.”

  14. Reactions that have a large DG in either direction are generally different for the forward vs reverse pathways.

  15. Glycolysis Reaction Step What is happening? *1) glucose + ATP --> G6P + ADP (hexokinase) 2) G6P --> F6P (phosphoglucoisomerase) *3) F6P + ATP --> F1,6 bisphosphate + ADP (phosphofructokinase) 4) F1,6bisP ---> G3P + DHAP (aldolase) 5) DHAP--> G3P (triosephosphate isomerase)

  16. Glycolysis Reaction Step What is happening? 6) G3P + NAD+ + Pi ---> 1,3 BPG + NADH (glyceraldehyde 3-phosphate dehydrogenase) 7) 1,3 BPG + ADP ---> 3 PG + ATP (phosphoglycerate kinase) 8) 3PG --> 2PG (phosphoglycerate mutase) 9) 2PG ----> PEP (enolase) *10) PEP + ADP ---> pyruvate + ATP (pyruvate kinase)

  17. Hexokinase has a regulatory domain as well as a catalytic domain To see more, click on the hexokinase pdb file link on the ISAT 350 home page.

  18. Hexokinase is inhibited by glucose 6 phosphate. Hexokinase is found throughout body. By contrast, glucokinase is only found in liver and is not inhibited by G-6-P.

  19. Km is measured in concentration units. The higher Km, the weaker the substrate binds. Typically, Km is close to the normal cellular concentration of the substrate.

  20. Feedback regulation: End products of a metabolic pathway can act as allosteric regulators of the initial steps of that pathway. A B C D

  21. phosphofructokinase F6P + ATP ------> F1,6 BP + ADP AMP + ATP - citrate - F2,6BP +

  22. F1,6 bisphosphatase F1,6 BP + H2O ----> F6P + Pi AMP - F2,6BP -

  23. How does allosteric regulation work? V S

  24. Sigmoidal Kinetics V S

  25. Proteins in RasMol and Chemscape Chime Ribbon view of pyruvate kinase (catalyzes the last step in glycolysis) In this view, the various colors correspond to individual subunit chains.

  26. Proteins in RasMol and Chemscape Chime Spacefilling view of triosephosphate isomerase In this view, red corresponds to regions with a helical structure, yellow regions are beta sheets and white regions are randomly structured regions.

  27. Protein Structure Review: Go to the link at Massey University http://www.massey.ac.nz/~wwbioch/Prot/tutehome/tutepage.htm Use their interactive tutorial on Protein Structure and Function to answer the question in “questions.doc”

  28. Proteins are:

  29. Sizes and Shapes of Proteins

  30. How Proteins Fold Proteins are composed of amino acids. Amino acids are linked by peptide bonds to form the primary structureof a protein. There are 20 different amino acids, each with unique side chains. The sequence of amino acids and the chemistry of the side chains determines how the protein folds which, in turn determines the protein structure and function.

  31. Side chains determine protein structure

  32. Nonpolar amino acids form a hydrophobic core hidden from water

  33. Secondary structure is stabilized by hydrogen bonds.

  34. Three types of noncovalent bonds stabilize protein folding.

  35. The alpha-helix Some proteins, such as cytochrome b are composed almost entirely of alpha-helices.

  36. The beta-sheet b sheets can be parallel or antiparallel

  37. The combination of a helices and b sheets constitute a protein’s secondary structure. The enzyme phosphoglucomutase from the glycolytic pathway.

  38. Proteins have several levels of organization Proteins can form higher levels of organization such as the coiled-coil of two alpha-helices shown. The three-dimensional conformation of a protein is referred to as the tertiary structure.

  39. Subunits Two or more polypeptide chains (subunits) can be joined to form a protein such as the CAP protein shown. When a protein has more than one polypeptide, the complete structure is designated the quaternary structure

  40. Disulfide bonds can covalently join two parts of the same protein or two different poylpeptides

  41. Protein Domains Different parts of a polypeptide chain can fold independently to form a stable structure called a domain. The different domains of a protein often have different functions such as the DNA binding domain (small) and the cyclic AMP binding domain of the CAP protein shown.

  42. Sizes of Proteins

  43. Review What types of noncovalent bonds help proteins fold? Name a covalent bond that stabilizes a protein’s three dimensional structure.

  44. The binding site is determined by amino acid side chains

  45. Antibodies selectively bind to antigens

  46. How do enzymes catalyze reactions?

  47. Lysozyme catalyzes the cutting of a polysaccharide chain

  48. The level and activities of an enzyme are regulated Gene expression can be regulated by the amount of substrate (the lac operon) Compartmentalization (proteases confined to the lysosome) Changes in conformations (allosteric changes) Protein phosphorylation

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