Lecture 7Outline of Previous Lecture1. Protein Function: Myoglobin & Hemoglobin2. Hemoglobin3. BPG Binding to HbOutline of Current Lecture1. Historical Background Catalysts as Protein Enzymes2. Enzyme Catalyzed Reactions3. Principles of Enzyme Active Site4. Equilibrium Point and Free Energy Changes are Related5. Acid-Base Catalysis6. ChymotrypsinCurrent Lecture1. Historical Background Catalysts as Protein EnzymesLate 1700s early 1800s –meat digestion by stomach secretions~1850 Vitalism – Louis Pasteur postulated that fermentation of sugar into alcohol by ferments that were inseparable from the cell1897 – Eduard Buchner – yeast extracts can ferment sugar and no cells neede: this advance marks the true beginning of biochemistryin many ways, Buchner “invented” biochemistry1926 – James Sumner—Crystallized an Enzyme: Urease. Enzymes made of protein (usually), Enzymes have distinctive & regular chemical structures2. Enzyme Catalyzed ReactionsE + S <-> ES <-> EP <-> E+PBasic formulation for single substrate reactionRegenerates free EEnzymes affect the rate of the reaction, but not its equilibrium positionEnzymes catalyze reactions in a bland chemical environment often near neutral pH, at mild temperatures, in aqueous solution, at modest pressure.Consider: S <-> P∆G’° - overall standard free energy change S  P at pH 7.0this simple description approximately applies to any reactionActivation Energy ∆G‡ is difference between energy levels of ground state and transition stateHigher ∆G‡ means a slower reactionOne can increase rate of reaction by increasing temperature – increasing the average kinetic energy of the molecules in the reaction or – by lowering the activation energyEnergy required for alignment of reacting groups, aslo involving partial bonds, bond breaking, rearrangements, forming unstable transient new bondsTransition state is a fleeting molecular state from which decay to either S or P ground state is equally probable and equally possibleWhat’s in an enzyme?Co-factor – “prosthetic group” – non-protein chemical component. Usually derived from vitaminsProteinMetal ion: Fe+2, Cu+2, Fe3+, K+, Mg+2, Mn+2, Ni+2, Zn+2, Mo, VaCoenzymes – complex organic or metallo-organic molecules transiet carries of specific functional groups permanently bound coenzymes are called prostheic groups. many coenzymes are derived from vitaminsApoprotein- protein without cofactors, coenzymes one subunitHoloenzyme – protein with cofactors, coenzymes, all subunits3. Principles of Enzyme Active SiteSite of catalysis—site where substrates are bound and converted to product specific for substrate and cofactors or coenzymes generally sequestered from solvent but has some solvent accessEnzyme specificity – substrate, cofactor, coenzyme specificity, stereospecificity, reaction specificityD-substrate won’t bind to an L-enzymeEnzyme Active Site principlesAllosteric Site or Modulator SiteSeparate site from active siteAffects catalysis directly or indirectly by modulating structureeffectors can up-regulate or down-regulate enzyme activityEnzyme Catalyzed ReactionsActive site of the enzyme – usually buried or sequestered from solution (but must be accessible!) lined with functional groups appropriate to interact with the substrateFunctional groups required for catalysis found here frequently a few bound and oriented water moleculesThe active site of the enzyme binds to the transition state of the substrate more tightly than to the groun state of S or P (don’t forget that products may be S and S may be products)Transition State S‡An activated form of the substrate or productCannot be isolated experimentallyNot called an intermediate (intermediates can be released and isolated)Has only very transient existenceWeak interactions between S‡ and active site optimized4. Equilibrium Point and Free Energy Changes are RelatedK’eq= [P]/[S]∆G’˚ = -RT lnK’eqthus the equilibrium constant K’eq is directly related to the net overall standard free energy change in the reaction ∆G’˚when K’eq is large, ∆G’˚ is more favorable – more negative)Reaction Rate/Velocity (mol/s)V = k[S1] FIRST ORDERV = k[S1][S2] SECOND ORDERK units are (s-1)Enzymatic Rates – from transition state theory, we can derive an equation that relates the rate constant to the activation energy5. Acid-Base CatalysisReaction accelerated by catalytic transfer of a protonSome AA R-groups in active site can accept and transfer protonsInvertase: A/B catalysisNever H+ donor or acceptor: Gly, Proline, Ala, Valine, Leucine, Isoleucine, Tryp, Phe, Gln, Asn6. Chymotrypsin – proteolytic enzyme – structure is 100% proteinOptimum pH = 8Pancreatic enzyme – must be secretedPre sequence  secretory pathway  Pro enzyme sequenceThree important residues: His 57, Ser 195, Asp 102 (don’t select aromatic side chains)His interacts with a Asp – stabilizes His destabilizes positive chargeSer – nuecleophileHis 57 must be deprotonated, ß-chain amino terminal +H3N-Ile16 must be protonatedCan’t always predict the AA involved because active site environment can shit R-group pH3 residues form a catalytic triad – key amino acids in the active site of chymotrypsinFavorite targets of ChymotrypsinPhe, Tyr, Tryp (bulky aromatic side chains – can fit in and be properly positioned to the backbone)Substrate Binding – polypeptide and phe/tyr make strong interactionsRecognition elements fit into binding pocketPositions peptide bond geometryHis borrows a proton from Ser – (Ser --OH  to –O-)Ser195 alkoxide ion – excellent nucleophileSer 195 lkoxide attacks peptide carbonyl, forms tetrahedral acyl-enzyme, negative charge on carbonyl is stabilized by H-bond in oxyanion holeH-bond from backbone amide of Ser 195Negative charge on carbonyl oxygen is unstable, so that electron pair moves to C-O bonding, thus double bond reforms to carbonyl carbon.Since C can never have 5 covalent bonds, C-N bond breaks. Breaking peptide bond requires amino leaving group be protonated by His57Product 1 leaves (carboxyl-terminal S fragment with new amino terminus)Fragment of S now attached to enzyme (N-terminal part linked as ester)Second chemical step: incoming water is deprontonated by His57 (general base catalyisis) strongly nucleophilic hydroxide ionHydroxide attacks ester carbon (reforms tetrahedral intermediate)Tetrahedral intermediate collapsesReformation of the carbonyl double bond breaks the acyl-enzyme