Binding and catalysis

1. Binding and catalysis Sherry Mowbray

2. Proteins have many functions • Many are enzymes: • Enzymes (20-40% of proteins, depending on organism) • Receptors • Transporters • Intracellular signaling • Structural role • Enzymes are catalytic proteins that convert their “substrates” into “products” • Virtually all protein functions require binding/recognition

3. Binding interactions • Ligand = something that binds reversiblyat a specific binding site; if it is acted on to change it chemically, we call it a “substrate”. • Ligands may be: • Small molecules e.g. catalysis (substrate) e.g. transport across a membrane (no chemical change) • Proteins of the same or different kind e.g. oligomers or multi-enzyme complexes e.g. signaling systems, etc

4. Molecules must touch (bind)to recognize each other • Usually noncovalent • Based on complementarity • Right size, shape • Right properties Which fits best?

5. Macromolecules are large So, complementarity means protein-protein interaction sites are usually: • on their surfaces • relatively flat • Exceptions with e.g. protein:DNA interactions (“grooves” are common, because DNA is long and thin) • relatively large (contact of 1500 Å2 or more) • affinity is roughly proportional to contact area

6. Interaction of THE SAME proteins • Many proteins are oligomers (dimers, trimers or such) for reasons of function or stability • May be possible to recognize surfaces • large, much non-polar character

7. Interaction of DIFFERENT proteins • Multienzyme complexes • E.g. RNA polymerase Bacterial is α2ββ’ω plus the associated sigma factor total of 420 kDa Often tight binding (can purify by His-tagging one chain) • May be possible to recognize • subunit-binding sites(often nonpolar) PDB: 3EQL

8. Interaction of DIFFERENT proteins • Some interactions are transient • binding is often weaker • sites often smaller in size • Harder to recognize • more like normal protein surface

9. Binding smaller molecules is different • Most enzymes work on small molecules • Protein folding creates pockets, clefts, tunnels in the surface of a protein • These are useful to bind smaller molecules • proteins don’t like holes, won’t do this without a reason • we can often recognize such sites, just from the structure • Between domains or subunits is common

10. Sixclassesofenzymes • Oxidoreductases (oxidation - reductionreactions; dehydrogenases) 2. Transferases

11. Sixclassesofenzymes, cont. 3. Hydrolases 4. Lyases

12. Sixclassesofenzymes, cont. 5. Isomerases 6. Ligases

13. A general reaction (definitions) • Transition state (TS), high energy arrangement of atoms and bonds needed to go from substrate (S) to product (P); has very high energy and so short lifetime (one vibration, 10-15 s). • DG, energy difference between S and P; reaction usually goes backwards, too (P usually is the one with lower energy) • Activation energy (DG‡ or Ea), energy needed to get from S (or P) to TS

14. Catalysis • Catalysis isn’t about doing the impossible, it is about making the possible happen faster • Catalyst (enzyme) doesn’t get changed itself • Doesn’t change DG or equilibrium constant between free S and P • Bicycle analogy

15. Reaction starts with substrate binding:complementarity! Each substrate has to dock • right charge might help attract the right things van der Waals interactions, hydrophobic interactions • mostly hydrophobic side chains • gives right shape, and energy Hydrogen bonds, electrostatic interactions • polar groups, mostly side chains, some main chain • gives energy and selectivity Kds: 10-3 to 10-6M, • i.e. not too tight, because that would make the hill higher

16. Catalysis can then occur:again, complementarity! • Protein have groups (usually side chains) IN THE RIGHT PLACE to • align the substrate(s) correctly • give the right chemistry (usually make/break bonds) • change as needed during the reaction

17. Catalysis means taking control of the environment • Chemical situations are created in a protein that don’t happen in solution (it is not magic, the same forces shape protein structure) • Like an auto workshop • shape, etc, is right to fit a car • and not a truck or motorcycle • they can close the doors • keep the rain (water) out • there is access to power • electricity for heat, lifts

18. Binding versus catalysis • Distinguish binding (specificity, Km) from catalysis (the reaction, kcat) • Often binding/catalytic sites are “separate” (don’t have to hold onto something that is changing) • protease example • Consider giving a dog a bath…

19. Common reasons for catalytic effects • Transition-state stabilization • Ground-state destabilization • Proximity effect

20. Transition-state stabilization • Tight binding stabilizes TS e.g. by placing groups that will bind to and stabilize a charge in the TS • Stable TS analogues bind tightly, are useful inhibitors

21. Ground-state destabilization • Example: chorismatemutase, where a less common (stable) form of substrate is bound • effectively raises ground state energy, gives a form ready for reaction • in the cell, [S] is generally below Km, binding is unfavorable, which has a similar effect

22. Proximity effect • Bring things close, in the right way • (think of two people being stuck in an elevator) • Example: ATCase, where there are no catalytic groups from the protein at all!

23. Multiple steps can simplify the problem (intermediates) Rather like taking your bicycle around the mountain, using another route, or simply getting off and walking it sometimes.

24. Other considerations • Conformational changes can be involved in a number of ways e.g. to close down on ligand, bring correct groups together e.g. to adjust during the reaction This will change kcat, Km… • Excluding water, or limiting it to one or more critical molecules, can be vital

25. The most common residues in active sites

26. Acid-base chemistry • Acids donate protons, and bases accept them • Virtually all enzymes do this in at least one step • pKasmust be accessible, and matched to the needs • Most popular are Glu, Asp, His (first two pKas4-5, His around 7) Glutamate Aspartate Histidine

27. pH affects enzymatic rates pH effects in reactions usually attributed to altered ionization of groups (side chains) in the active site BUT change in pH may also alter substrate binding/product release or 3D structure of enzyme. Can assay enzymatic activity at different pHs, plot activity vs pH Inflection point -> approximation of pKa of that ionizable group

28. Taking control… Enzymes can place groups in an environment that will alter their pKa • e.g. if you put an acid in a hydrophobic environment, this will favor uncharged form • e.g. put two charged residues close together, they will share a proton, or otherwise affect each other’s pKa Changes of pKa by 2-3 units are common

29. Nucleophiles/electrophiles Two types of ionic species common in active sites • Nucleophilic, electron-rich (negatively charged or having free, unshared electron pair) • Electrophilic, electron-poor, positively charged Nucleophilic attack on an electrophilic species is part of many reactions, leading to substitution or displacement Most common nucleophiles in proteins are Ser and Cys electrophile nucleophile

30. When the 20 amino acids aren’t enough… Cofactor: non-protein component of many enzymes needed for activity. Can be a metal ion (commonly Fe, Mg, Zn, Cu). Cofactors take direct part in the catalyzed reaction, and may be modified during it. If so, they may require another enzyme-catalyzed reaction to return them to the original state.Cofactors are often bound loosely to an enzyme and can be easily separated from it. Coenzyme: an organic cofactor; usually transfer something to a substrate hydride, electrons, small carbon-containing units (often things that are unstable or toxic); frequent in redox reactions. Prosthetic group: a coenzyme that is firmly bound to the apoenzyme and cannot be removed without denaturing it, or breaking a covalent bond; most contain a metal such as copper or iron Holoenzyme: for an enzyme that needs a cofactor, the complete, active enzyme Apoenzyme: for an enzyme that needs a cofactor, the empty inactive form, which may not fold stably.(We also frequently refer to any empty enzyme as the apo form.) Humans get most cofactors from vitamins and minerals in their diet.

31. Example: a serine protease, chymotrypsin Serine proteases catalyse the hydrolysis of peptides Catalytic triad (chymotrypsin) - is this reaction sensitive to pH? What residue in the catalytic triad do you think is most important?

33. The reaction for the lab protein(spinach RpiA) Ribose-5-P <=> ribulose-5-P no metal requirements are known As for most isomerases, the reaction is in equilibrium. Necessary to use ribose in metabolism making new sugar and recycling old

34. Step 1: ring opening Step 2: isomerization

35. One structure, various functions • Example: TIM barrel • An extremely common fold • Active sites always at C-terminal end of barrel (sheet) • May do same reaction on a different substrate • But many, many other reactions can be catalyzed • Often, but not always metalloenzymes • Another example: 7TM proteins • Many but not all are GPCRs

36. One function, various structures • Example: ribose-5-phosphate isomerase • Two kinds, with the same steps in the mechanism • Completely different amino acids involved RpiA (most organisms) RpiB (e.g. some bacteria) lab protein