Catalytic StrategiesTwo Substrate Reactions (Sequential: when two or more substrates bind to one enzyme)Ordered Sequential: all reactants/substrates are bound to the enzyme in a defined order or sequenceEx: lactate dehydrogenase enzymeRandom: substrates are bound and released in no particular orderEx: creatine kinase enzymePing pong (aka double displacement reaction) (nonsequential)Enzyme bounces back and forth from an intermediate state to its standard stateOne product is formed and released before the second substrate bindshttp://chemwiki.ucdavis.edu/Biological_Chemistry/Catalysts/Enzymatic_Kinetics/Ping-pong_mechanismsEnzymesLower activation energy of the reactionIf we think of S as being in equilibrium with the transition state (X+), then the smaller of difference between free energy and S and X+, then there is a greater concentration of X+Tight bindingAn enzyme with a pocket complimentary to the transition state helps to destabilize the substrate, thereby contributing to catalysisCatalytic Mechanisms of EnzymesProximity and Orientation EffectsComplex III: respiratory chain enzyme (do we need to know mechanism for Complex III)Cytochrome c (substrate) binds to the subunit cytochrome c1 and preorientation of these protein partners is required for heme to heme electron transferCatalysis by Preferentially Binding the Transition StateCatalytic Triad of Serin ProteasesHis 57, Asp 102, Ser 195, Specificity Pocket NAfter the protease has bound a protein substrate,Ser 195 nucleophillically attacks the scissile peptide’s carbonyl group to form the tetrahedral intermediateOxyanion hole: a hole inside of the serin protease that stabilizes a deprotonated oxygen or alkoxideWithout the oxyanion hole the oxyanion (carboxyl oxygen) hydrogen binds to the NH group of Gly 193 and the NH group of Ser 195. This conformation also allows the NH group of the preceding amino acid (Rattached to the trigonal carbon atom) to hydrogen bond to the C=O group of Gly 193 =STRONGER BINDINGEvidence for the existence of the tetrahedral intermediate: X-ray structure of trypsin and its inhibitor BPTIBPTI: inhibits the normal proteaolysis reaction closely associated with the preceding tetrahedral transition stateTryptin has Ser 195 and whose oxygen nucleophilically attacks the trigonal carbon atom. BPTI instead binds to that oxygen with its carbon so that the Ser195 can’t bind its oxygen to anything elseCatalysis by Substrate DestabilizationAnticataytic: An increase in activation energy results from the ES having less free energy in the ground state than the substrate alone “magnets” in enzymeDestabilization: can facilitate catalysis if:1. The substrate energy is brought closer to X+ (transition state)2. Destab is relieved at X+Destab: raises the free energy of the substrate bringing it closer to the X+, destab of ES facilitates catalysisEntropy reduction (destabilization): substrate becomes more reactive because its free energy is increased by its decrease in available degrees of freedomAlone the enzyme and the substrate are in a disordered high entropy state> translational motionDesolvation (Destabilization): water is more reactive because its free energy is raised from removing a very polar functional group, “solvation shell” around the substrate before it binds with enzymeSubstrate is given a positive chargeStress (Destabilization): electrostatic or stericSubstrate is more reactive because its free energy is raised from the juxtaposition of 2 like charges withinthe substrateAcid Base CatalysisSpecific: involves OH or H+ diffusing into the catalytic centerGeneral: involves acids other than H+ and bases other than OHThese other acids and bases facilitate transfer of H+ in the transition stateGeneral acid catalysis proton transfer from the acid to the substrate lowers the ΔG of the transition stateGeneral base catalysis: proton is abstracted by the base K increases as buffer is added RNase AHis 12 acts as base by abstracting a proton from RNZ 2’ OH therefore promoting its nucleophillic attack on the phosphateHis 119 acts as an acid promotes bond scission by protonating the leaving nucleotideChymotrypsin: ping ping kinetics, nucleophillic catalysis, stabilization of the transition state, general acid base catalysis, Asp 102, His 57, Ser 195Substrate specificity of chymotrypsin: cleavage occurs only on the C terminal side of aromatic and large hydrophobic residuesCatalytic triad of serin proteases: chymotrypsin, trypsin, elastase, subtilisin Specificity pocket of chymotrypsin: Ser 189, aromatic Phenol ring, Gly 226, Gly 216Specificity pocket of Tyrpsin: Asp 189, Lys, Gly 226, Gly 216Specificity pocket of Elastase: Val 226, Thr 216, AlaCatalyzed reaction of chymotrypsin:Favorable ΔG but very high transition state free energy***Memorize and practice steps and details of Chymotrypsin reaction mechanismDIPF: inhibitory molecule that provides evidence for nucleophillic catalysis in the chymotrypsin reaction mechanism Burst Kinetics (Burst Phase): point in enzyme kinetics that refers to initial high velocity of product formation and enzyme turnoverSteady state phase (Burst kinetics): once all the enzymes are saturated with substrate, linear velocity takes place Cysteine proteases: Cys is nucleophile, Histidine is the general base catalyst Regulatory strategies for proteases: Pancreatic trypsin inhibition: Lys 59, Asp 189Zymogen: an enzyme before it becomes active; becomes active through proteolysis when it is cleaved at specific sites so that I can change its shapeERGolgiZymogen lumen Trypsinogen: zymogen of trypsin; pancreasEnteropeptidase: protease that cleaves trypsinogen which leads to a proteolysisTrypsin: cleaves proelastaseelastase (pancreas) ; cleaves procarboxyepetidase  carboxypeptidase (pancreas), chymotrypsinogeinchymotrypsin (pancreas); prolipaselipaseThrombin: first step in the clotting processFibrin: converted form of fibrinogen; when the ends (tails) are cleaved off, the active site is exposed so that it can bond with other fibrins to form clots, active sites from removal of the top tails are Gly-His-Arg sequences, active sites from removal of bottom tails are Gly-Pro-Arg SeuencesTransglutaminase: binds Gly of one fibrin to Lys of another by forming a cross link and removing NH4+(ammonium)Intrinsic pathway: begins with a damaged surface, kininogen kallikrein system (poorly understood hormonal system with limited available research)Extrinsic pathway: begins with