Protein Functions; Enzyme Properties

1. Protein Functions;Enzyme Properties Andy HowardIntroductory Biochemistry, Fall 2009 15 September 2009 Biochem: Protein Functions I

2. Proteins and enzymes • Proteins perform a variety of functions, including acting as enzymes. Biochem: Protein Functions I

3. Visualizing structure The Protein Data Bank Tertiary & quaternary structure Protein Functions Structure-function relationships Protein Functions PTM Allostery Classes of proteins and their roles Enzyme properties Classes of enzymes Enzyme kinetics Plans for Today Biochem: Protein Functions I

4. How do we visualize protein structures? • It’s often as important to decide what to omit as it is to decide what to include • Any segment larger than about 10Å needs to be simplified if you want to understand it • What you omit depends on what you want to emphasize Biochem: Protein Functions I

5. Styles of protein depiction • All atoms • All non-H atoms • Main-chain (backbone) only • One dot per residue (typically at C) • Ribbon diagrams: • Helical ribbon for helix • Flat ribbon for strand • Thin string for coil Biochem: Protein Functions I

6. How do we show 3-D? • Stereo pairs • Rely on the way the brain processes left- and right-eye images • If we allow our eyes to go slightly wall-eyed or crossed, the image appears three-dimensional • Dynamics: rotation of flat image • Perspective (hooray, Renaissance) Biochem: Protein Functions I

7. Straightforward example • Sso7d bound to DNAGao et al (1998) NSB 5: 782 Biochem: Protein Functions I

8. A little more complex: • Aligning Cytochrome C5with Cytochrome C550 Biochem: Protein Functions I

9. Stereo pair: Release factor 2/3Klaholz et al, Nature (2004) 427:862 Biochem: Protein Functions I

10. Mostly helical:E.coli RecG - DNA PDB 1gm53.24Å, 105 kDa Mixed:hen egg-white lysozyme PDB 2vb10.65Å, 14.2kDa Ribbon diagrams Biochem: Protein Functions I

11. The Protein Data Bank • http://www.rcsb.org/ • This is an electronic repository for three-dimensional structural information of polypeptides and polynucleotides • 55660 structures as of 1pm today • Most are determined by X-ray crystallography • Smaller number are high-field NMR structures • A few calculated structures, most of which are either close relatives of experimental structures or else they’re small, all-alpha-helical proteins Biochem: Protein Functions I

12. What you can do with the PDB • Display structures • Look up specific coordinates • Run clever software that compares and synthesizes the knowledge contained there • Use it as a source for determining additional structures Biochem: Protein Functions I

13. Generalizations about Tertiary Structure • Most globular proteins contain substantial quantities of secondary structure • The non-secondary segments are usually short; few knots or twists • Most proteins fold into low-energy structures—either the lowest or at least in a significant local minimum of energy • Generally the solvent-accessible surface area of a correctly folded protein is small Biochem: Protein Functions I

14. Hydrophobic in, -philic out • Aqueous proteins arrange themselves so that polar groups are solvent-accessible and apolar groups are not • The energetics of protein folding are strongly driven by this hydrophobic in, hydrophobic out effect • Exceptions are membrane proteins Biochem: Protein Functions I

15. Domains • Proteins (including single-polypeptide proteins) often contain roughly self-contained domains • Domains often separated by linkers • Linkers sometimes flexible or extended or both • Cf. fig. 6.36 in G&G Biochem: Protein Functions I

16. Generalizations about quaternary structure • Considerable symmetry in many quaternary structure patterns(see G&G section 6.5) • Weak polar and solvent-exclusion forces add up to provide driving force for association • Many quaternary structures are necessary to function:often the monomer can’t do it on its own Biochem: Protein Functions I

17. Protein Function: Generalities • Proteins do a lot of different things. Why? • Well, they’re coded for by the ribosomal factories • … But that just backs us up to the question of why the ribosomal mechanism codes for proteins and not something else! Biochem: Protein Functions I

18. Proteins are chemically nimble • The chemistry of proteins is flexible • Protein side chains can participate in many interesting reactions • Even main-chain atoms can play roles in certain circumstances. • Wide range of hydrophobicity available (from highly water-hating to highly water-loving) within and around proteins gives them versatility that a more unambiguously hydrophilic species (like RNA) or a distinctly hydrophobic species (like a triglyceride) would not be able to acquire. Biochem: Protein Functions I

19. Structure-function relationships • Proteins with known function: structure can tell is how it does its job • Example: yeast alcohol dehydrogenase:Catalyzesethanol + NAD+ acetaldehyde + NADH + H+ • We can say something general about the protein and the reaction it catalyzes without knowing anything about its structure • But a structural understanding should help us elucidate its catalytic mechanism Biochem: Protein Functions I

20. Why this example? • Structures of ADH from several eukaryotic and prokaryotic organisms already known • Yeast ADH is clearly important and heavily studied, but until 2006: no structure! • We got crystals 8 years ago, but so far I haven’t been able to determine the structure Yeast ADH PDB 2hcy2.44Å 152 kDa tetramerdimer shown Biochem: Protein Functions I

21. What we know about this enzyme • Cell contains an enzyme that interconverts ethanol and acetaldehyde, using NAD as the oxidizing agent (or NADH as the reducing agent) • We can call it alcohol dehydrogenase or acetaldehyde reductase; in this instance the former name is more common, but that’s fairly arbitrary (contrast with DHFR) Biochem: Protein Functions I

22. Size and composition • Tetramer of identical polypeptides • Total molecular mass = 152 kDa • We can do arithmetic: the individual polypeptides have a molecular mass of 38 kDa (347 aa). • Human is a bit bigger: 374 aa per subunit • Each subunit has an NAD-binding Rossmann fold over part of its structure Biochem: Protein Functions I

23. Structure-functionrelationships II • Protein with unknown function: structure might tell us what the function is! • Generally we accomplish this by recognizing structural similarity to another protein whose function is known • Sometimes we get lucky: we can figure it out by binding of a characteristic cofactor Biochem: Protein Functions I

24. What proteins can do: I • Proteins can act as catalysts, transporters, scaffolds, signals, or fuel in watery or greasy environments, and can move back and forth between hydrophilic and hydrophobic situations. Biochem: Protein Functions I

25. What proteins can do: II • Furthermore, proteins can operate either in solution, where their locations are undefined within a cell, or anchored to a membrane. • Membrane binding keeps them in place. • Function may occur within membrane or in an aqueous medium adjacent to the membrane Biochem: Protein Functions I

26. What proteins can do: III • Proteins can readily bind organic, metallic, or organometallic ligands called cofactors. These extend the functionality of proteins well beyond the chemical nimbleness that polypeptides by themselves can accomplish • We’ll study these cofactors in detail in chapter 17 Biochem: Protein Functions I

27. Zymogens and PTM • Many proteins are synthesized on the ribosome in an inactive form, viz. as a zymogen • The conversions that alter the ribosomally encoded protein into its active form is an instance of post-translational modification PDB 3CNQSubtilisin prosegment complexed with subtilisin1.71Å; 35 kDa monomer Biochem: Protein Functions I

28. Why PTM? • This happens for several reasons • Active protein needs to bind cofactors, ions, carbohydrates, and other species • Active protein might be dangerous at the ribosome, so it’s created in inactive form and activated elsewhere • Proteases (proteins that hydrolyze peptide bonds) are examples of this phenomenon • … but there are others Biochem: Protein Functions I

29. Protein Phosphorylation • Most common form of PTM that affects just one amino acid at a time • Generally involves phosphorylating side chains of specific polar amino acids:mostly S,T,Y,H (and D, E) • Enzymes that phosphorylate proteins are protein kinases and are ATP or GTP dependent • Enzymes that remove phosphates are phosphatases and are ATP and GTP independent Biochem: Protein Functions I

30. iClicker question 1 Why are digestive proteases usually synthesized as inactive zymogens? • (a) Because they are produced in one organ and used elsewhere • (b) Because that allows the active form to be smaller than the ribosomally encoded form • (c) To allow for gene amplification and diversity • (d) So that the protease doesn’t digest itself prior to performing its intended digestive function • (e) None of the above Biochem: Protein Functions I

31. iClicker question 2 Which amino acids can be readily phosphorylated by kinases? • (a) asp, phe, gly, leu • (b) ser, thr, tyr, his • (c) leu, ile, val, phe • (d) arg, lys, gln, asn • (e) none of the above. Biochem: Protein Functions I

32. iClicker question 3 Why are kinase reactions ATP- (or GTP-) dependent, whereas phosphatase reactions are not? • (a) To ensure stereospecific addition of phosphate to the target • (b) To prevent wasteful hydrolysis of product • (c) Adding phosphate is endergonic; taking phosphate off is exergonic • (d) None of the above. Biochem: Protein Functions I

33. Allostery • Formal definition:alterations in protein function that occur when the structure changes upon binding of small molecules • In practice: often the allosteric effector is the same species as the substrate: they’re homotropic effectors • … but not always: allostery becomes an effective way of characterizing third-party (heterotropic) activators and inhibitors Biochem: Protein Functions I

34. v0 What allostery means [S] • Non-enzymatic proteins can be allosteric:hemoglobin’s affinity for O2 is influenced by the binding of O2 to other subunits • In enzymes: non-Michaelis-Menten kinetics (often sigmoidal) when the allosteric activator is also the substrate Biochem: Protein Functions I

35. R and T states • Protein with multiple substrate binding sites is in T (“tense”) state in absence of ligand or substrate • Binding of ligand or substrate moves enzyme into R (“relaxed”) state where its affinity for substrate at other sites is higher Glycogen phosphorylase BPDB 1XC7 98 kDa monomer1.83Å Biochem: Protein Functions I

36. R and T state kinetics • Binding affinity or enzymatic velocity can then rise rapidly as function of [S] • Once all the protein is converted to R state, ordinary hyperbolic kinetics take over Biochem: Protein Functions I

37. Other effectors can influence RT transitions • Post-translational covalent modifiers often influence RT equilibrium • Phosphorylation can stabilize either the R or T state • Binding of downstream products can inhibit TR transition • Binding of alternative metabolites can stabilize R state Biochem: Protein Functions I

38. Why does that make sense? • Suppose reactions are: (E)A  B  C  D • Binding D to enzyme E (the enzyme that converts A to B) will destabilize its R state, limiting conversion of A to B and (ultimately) reducing / stabilizing [D]: homeostasis! Biochem: Protein Functions I

39. Alternative pathways • Often one metabolite has two possible fates: B  C  DA H  I  J • If we have a lot of J around, it will bind to the enzyme that converts A to B and activate it; that will balance D with J! Biochem: Protein Functions I

40. How does this work structurally? • In general, binding of the allosteric effector causes a medium-sized (~2-5Å) shift in the conformation of the protein • This in turn alters its properties • Affinity for the ligand • Flexibility (R vs T) • Other properties • We’ll revisit this when we do enzymology Biochem: Protein Functions I