Ch395G Fall 2009: Enzyme CatalysisEnzymatic CatalysisVoet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Enzymatic Catalysis1. Short Review: What is an Enzyme?DefinitionTransition State Theory2. What are the chemical mechanisms for accomplishing catalysis?Preferential Binding of the Transition StateProximity and Orientation EffectsAcid – Base Catatysis keto-enol tautomerization Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Ribonuclease ACovalent Catalysis (PLP-, CoA-SH, THF, TPP-dependent enzymes)Metal Ion Catalysis 3. Lysozyme as an example – look for how rate is enhanced and the methods used to determine mechanistic information.Biochemical DataStructural DataPhillips MechanismRecent DevelopmentsMetal Ion CatalysisMetalloenzymesMetal-activated enzymesVoet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Metal ions can participate in catalysis in 3 different ways : a) orienting substrate b) mediating redox reactions c) stabilizing negative chargeHEW Lysozyme:14.7 kDa, 129 a.a.4 -S-S-kcat/kuncat = 10x8Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Figure 15-9 Primary structure of HEW lysozyme.Page 507First EnzymeStructure - 1965Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Figure 15-8 The alternating NAG–NAM polysaccharide component of bacterial cell walls.Page 507Biochemical DataTable 15-2 Rates of HEW Lysozyme-Catalyzed Hydrolysis of Selected Oligosaccharide Substrate Analogs.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Page 508Sequence Alignments: Conserved residues – E35, D52X-ray DataVoet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Page 509Figure 15-10b X-Ray structure of HEW lysozyme. (b) A ribbon diagram of lysozyme highlighting the protein’s secondary structure, (c) A computer-generated model showing the protein’s molecular envelope (purple) and Cαbackbone (blue).X-ray DataVoet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Page 509Figure 15-10a X-Ray structure of HEW lysozyme. (a) The polypeptide chain is shown with a bound (NAG)6substrate (green).See the Chapter 15 Interactive Exercise 3 about lysozyme on the Voet & Voet CD. Spend some time with the 3D model making sure that you can manipulate the program to rotate Using 3D ModelsVoet Biochemistry 3e© 2004 John Wiley & Sons, Inc.that you can manipulate the program to rotate lysozyme, zoom in and out, change from ball and stick representation to ribbon and to space filling depictions.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Page 510Figure 15-12 Interactions of lysozyme with its substrate.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Figure 15-11 Chair and half-chair conformations.Page 510___Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Table 15-3 Binding Free Energies of HEW Lysozyme Subsites.Page 513+__From their proximity to the cleavage site of the glycosidic bond betweensubstrate D and E rings Asp52 and Glu 35 were postulated to bethe primary catalytic residues. The difference in environments allows the 2 acidic side chains to have very Figure 15-14 The Phillips mechanism for the lysozyme reaction.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.2 acidic side chains to have very different effective pKa values. Asp 52 is predicted to have a normal pKa and be unprotonated (that is, negatively charged) throughout the enzymatic process. It is thought to stabilize the oxonium ion formed during the reaction. Glu 35 is thought to be the proton donor for the reaction (general acid catalyst). See Fig. 15-14 for an overview of the mechanism.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Figure 15-13 Mechanism of the nonenzymatic acid-catalyzed hydrolysis of an acetal to a hemiacetal.Page 511Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Figure 15-15 The D-ring oxonium ion intermediate in the Phillips mechanism is stabilized by resonance.Page 512NAG lactone inhibitor binds to D-subsitewith 9.2 kJ/mol greater affinity (~ 40x).Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Figure 15-16 The δ-lactone analog of (NAG)4.Page 513But !!! ……Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Figure 15-17 The HEW lysozyme covalent intermediate.Page 515Summary –What “chemical” mechanisms were employed in Lysozyme?Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.What methods provided the data to work out the current mechanism?Control of Enzymatic ActivityRates of synthesis and degradation (Nobel Prize for the ubiquitination pathway)See Chapter 13 pages 465 - 470Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.for the ubiquitination pathway)Proenzyme (zymogen) conversionPresence of activators and inhibitorsCovalent ModificationAllosteric ControlFrom the NationalVoet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Ubiquitin tagging of proteins for degradationIrwin Rose (UCSD) one of the 3 Nobel Prize winnersVoet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Enzyme Control by Covalent ModificationEnzymes can either by activated or deactivated by phosphorylation.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Phosphorylation sites include tyrosine, serine and threonineVoet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Allosteric Control of Enzymatic ActivitySee Chapter 10 pp 345-353 for introduction to this topicTwo Different Theoretical ModelsVoet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Two Different Theoretical ModelsSymmetry Model (Monod, Wyman, Changeux 1965)Sequential Model(Koshland 1966)This material was not covered in depth in the lectures and questions will only be asked in exams to the level it was covered in class.Symmetry ModelVoet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Symmetry ModelVoet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Figure 10-29 The species and reactions permitted under the symmetry model of allosterism.Page 347Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Figure 10-30 Symmetry model saturation function curves for tetramers according to Eq. [10.22].Page 348Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Figure 10-31 Heterotropic interactions in the symmetry model of allosterism.Page 349Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Figure 10-32 The effects of allosteric activator (γ = [A]/kA) and