Mechanisms of Enzyme Action Catalytic Mechanisms 1 Enzymes as Catalysts Effectively lower the free energy of the transition state G by stabilizing the transition state of the catalyzed mechanism Promote orientation of substrate s within the enzyme active site to stabilize the transition state thus promoting the reaction 2 Enzymes as Catalysts Enzymatic mechanisms are similar to nonenzymatic reactions Importance of functional groups and a fundamental understanding of organic chemistry Curved arrow convention trace electron pair rearrangements where electrons are and the path to where they are going e deficient e deficient e rich e rich Never 5 bonds to a carbon or 2 bonds to a hydrogen3 Enzyme Catalysis Three basic types of catalytic mechanisms 1 Covalent catalysis 2 Acid base catalysis 3 Metal ion catalysis Know 3 basic types of catalytic mechanisms and understand each 4 1 Covalent Catalysis Accelerates reaction rates through the transient formation of a catalyst substrate covalent bond Bond usually formed between a nucleophilic e rich group on enzyme and electrophilic e deficient group on the substrate Catalyst substrate covalent bond 5 2 Acid Base Catalysis Most enzyme reactions involve some degree of acid or base catalysis 1 Specific acid base catalysis reaction is accelerate by H or OH diffusing in from the solution 2 General acid base catalysis catalysis in which a proton is transferred in the transition state 6 2 Acid Base Catalysis Concerted reactions Acid catalyzed Base catalyzed PROTON TRANSFER General acid catalysis A process in which a proton transfer from an acid lowers the free energy of a reaction s transition state General base catalysis A process in which a proton abstraction by a base lowers the free energy of a reaction s transition state Side chains of amino acids make good acids and bases Asp Glu His Cys Tyr and Lys 7 How to determine which residues involved in acid base catalysis pH profile Assesses the effect pH has on the rate of the enzymatic reaction Allows for identification of the pKa of the ionizable residue s How does this work 3 possibilities e t a R e t a R e t a R Increase in activity at higher pH H off Residue must be deprotonated for optimal catalytic activity Increase in activity at lower pH H on Residue must be protonated for optimal catalytic activity Bell shaped curve 2 residues One residue must be protonated and another residue deprotonated for optimal catalytic activity 8 5 6 7 8 9 pH 5 6 7 8 9 pH 5 6 7 8 9 pH RNase A Acid Base Catalyst by the pancreas Digestive enzyme secreted Hydrolyzes RNA to its component nucleotides Contains two ionizable residues Both His residues His12 acts as a base His119 acts as an acid pH profile Sample Test Question Involving pH profiles Mechanism of RNase A 9 pH profile pH optimum Enzymes possess ionizable side chains that have roles in 1 structure formation and 2 involved in the active site Enzymes are active only over a limited pH range Most have a particular pH in which the catalytic activity is optimal Study of enzyme s pH profile help distinguish the pH optimum for the enzyme 10 Figure 13 11 p422 3 Metal Ion Catalysis Nearly 1 3 of all known enzymes require the presence of metals for catalytic activity Metalloenzymes contain tightly bound metal cofactors such as Fe2 Fe3 Cu2 Zn2 Mn2 Co2 Metal Activated enzymes only loosely bind the metal ions The ions are usually Na K Mg2 or Ca2 and usually play a structural role 11 Metal Ion Cofactors 3 ways metal ions play a role in catalysis 1 Binding to substrates for orientation 2 Mediation of redox rxns by changing oxidation state 3 Stabilizing or shielding negative charges Example Carbonic Anhydrase Essential Zn2 ion Why Binds a water molecule that is necessary for catalysis making it more acidic donates H thus allowing for formation of the nucleophile OH Im Histidine imidazole ring side chain 12 Catalytic Mechanisms Serine Proteases active site Ser 1 Trypsin 2 Chymotrypsin 3 Elastase Important Reminders about Proteases 1 Cleave scissile bonds through a hydrolysis reaction therefore 2 are hydrolases Aspartic Proteases active site Asp 1 HIV 1 Protease 13 Mechanism of Serine Proteases General Base Nucleoph ile The Catalytic Triad The active sites of trypsin chymotrypsin and elastase all are composed of a catalytic triad His Asp and Ser Figure 14 17 p464 H bond orients His 14 Figure 11 29 15 Ser195 attacks the scissile bond through nucleophilic attack Proton is transferred to the His57 imidazole ring aided by the polarizing effect of Asp102 Figure 11 29 part 1 16 Tetrahedral intermediate decomposes to the acyl enzyme intermediate Proton from His57 is donated from N3 to stabilize acyl enzyme intermediate Figure 11 29 part 2 17 Acyl enzyme intermediate is subject to hydrolysis Amine leaving group is replaced by H2O Figure 11 29 part 3 18 H2O acts as a nucleophile and attacks the carbonyl of the acyl enzyme intermediate forming a second tetrahedral intermediate Figure 11 29 part 4 19 A reversal of step 1 yields the carboxylate product the new C term portion of the cleaved polypeptide chain Note that active site of the serine protease is ready for the next round of catalysis Figure 11 29 part 5 20 Figure 11 29 21 Transition State Analogs Serine proteases are examples of enzymes that preferentially bind the transition state The tighter an enzyme binds the transition state the greater the rate of the catalyzed reaction relative to the uncatalyzed reaction Conformational distortions accompanying formation of tetrahedral intermediate cause the carbonyl oxygen to move deeper into active site oxyanion hole 22 Oxyanion Hole Conformational distortions accompanying formation of tetrahedral intermediate cause the carbonyl oxygen to move deeper into active site oxyanion hole The preferential binding of the transition state or the tetrahedral intermediate over the enzyme substrate complex or the acyl intermediate is responsible for much of the catalytic efficiency of serine proteases 23 Thermodynamic Explanation The oxyanion hole serves to lower the transition state free energy for the formation of the tetrahedral intermediate Td 1 Td 2 Acyl E I 24 Aspartic Proteases Aspartic proteases are a family of proteases that have two active site aspartic acids residues These enzymes are active at acidic pHs Aspartic proteases are important in digestion pepsin and regulation of blood pressure Two Asp form catalytic dyad a b extraction of 2 protons Asp32 C O leads to nucleophilic attack c d