Ch11-120309 - CHEM 350: Introduction to Biological...

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Unformatted text preview: CHEM 350: Introduction to Biological Chemistry Brian Lee, Ph.D. [email protected] Office: Neckers 146G or 324 Phone: 453-7186 Ho urs: 9:30am to 10:30am or by appointment Website: https:/ / Textbook (required, U.S. edition only) Fundamentals of Biochemistry, 3rd Ed., Voet, Voet & Pratt. Study Guide (recommended) Student Companion to Fundamentals of Biochemistry, 3rd Ed. Help Desk Tuesday 6:30 to 7:30 pm in Neckers 218 Thursday 5:00 to 6:00 pm in Neckers 410 Announcements Undergraduate Research Opportunities Research for credit (such as CHEM 396 or CHEM 496) Student worker ($8.00 per hour) ( Undergraduate Assistantships ( McNair Scholars Program ( REACH Awards Competition ( Assignments Read Chapter 12 Enzyme Kinetics Chapter 12 Problems Third Midterm Exam, Wednesday March 28th Chapters 10-13 Catalytic Mechanisms • Acid-base catalysis • Covalent catalysis • Metal-ion catalysis • Electrostatic catalysis • Proximity and orientation • Transition state binding • Acid-base catalysis –general acid catalysis involves partial proton transfer from an acid group which lowers the free energy of the transition state –general base catalysis involves partial proton abstraction by a basic group which lowers the free energy of the transition state –pH dependence Example: keto-enol tautomerization uncatalyzed acid catalyst base catalyst Acid-Base catalysis of ester bond. Amide is a potential leaving group if protonated. How can an enzyme promote the formation of product? Protonation of amide group – potential for catalysis Uncatalysed Reaction Water donates proton No option to improve rate by acid-base catalysis 1) exclude water from active site Catalysed Reaction Potential to enhance reaction rate by acid-base catalysis. HA donates proton. B: accepts proton. 2) position of acidic functional group Transition state stabilized by moving the proton from the ester bond to the amide bond. The protonated amide group is now an excellent leaving group. Restoration of Enzyme Protonated product will donate proton to the enzyme directly or indirectly via water pH dependence defined by pKa of acid-base functional groups required for catalysis RNase A His119 His12 Mechanism for RNAse A Histidine pKa 6.0 His12 acts as a base c His119 acts as an acid catalyst (higher pKa) The pKa of a particular amino acid residue can vary depending on local chemical environment. • Covalent catalysis (nucleophilic catalysis) –the transient formation of a covalent bond between enzyme and substrate stabilizes the transition state thus accelerating the rate of reaction nucleophilic attack by the primary amine on the electrophilic carbonyl carbon electron rearrangement to form the Schiff base the carbon atom of the imine is a good electrophile Page 334 Decarboxylation of acetoacetate - primary amine catalyst (an unprotonated lysine) - hydroxyl exchanged with water The enolate intermediate is stabilized by formation of a Schiff base Bases or the conjugate base of a very weak acids can form good nucleophiles after losing a proton. Acidic protons, metal ions, carbon atoms in carbonyls and cationic imines are good electrophiles serine or aspartate cysteine carbanion TPP (thiamine pyrophosphate) lysine histidine water peptide bond Schiff base imine (pyridoxal phosphate) phosphorus ATP or GTP water • Metal-ion catalysis – metal ion required for catalysis • metalloenzymes – metal required for catalysis tighty bound metal ion (usually transition metal ions): Fe2+, Fe3+, Cu2+, Zn2+, Mg2+, Mn2+, Co2+ • metal-activated enzymes – structural role loosely bind metal ions from solution (usually alkali and alkaline earth metals): Na+, K+, Zn2+, Mg2+, Ca2+ – catalytic mechanisms • substrate binding and orientation • oxidation-reduction • electrostatic interaction Carbonic anhydrase Zinc activates a water molecule to create a nucleophilic OH- nucleophilic attack exchange with water reactivation of water bicarbonate • Electrostatic catalysis –the charge distribution around the active site is arranged to stabilize the transition state –active sites generally exclude water, so the local dielectric constant within the active site will enhance electrostatic interactions Enolase requires two Mg2+ ions to stabilize the negative charge on the carboxylate group Lys345 acts as a general base and Glu211 as a general acid to catalyze to removal of a hydroxyl group. The Mg2+ ions stabilize two negative charges in the enolic intermediate. • Proximity and orientation –two substrates are bound by the enzyme in close proximity and in the optimal orientation for catalysis –there is minimal enhancement from proximity alone Rate enhancement by entropy reduction ester carboxylate proximity and orientation better proximity and orientation anhydride • Transition state binding –preferential binding to the transition state lowers the free energy of the transition state and increases the rate of reaction –transition state analogs make powerful inhibitors of enzymes Binding energy ( GB) reduces the energy barrier. The transition state binding is favored by enthalpy. Binding energy ( GB) can directly reducing the G The reduction in G due to catalysis is G cat Transition State Theory V = kbT/h [A] e- G‡/RT For a 10 fold rate enhancement with V’ = 10 x V V ' k b T h [ A] e = V k b T h [ A] e G RT G RT 10 = e( G*' G* ln(10) = 2.303 = = RT G cat G RT ) ( * Gcat RT = 5.9 kJ/mol at 37°C Hydrogen bond enthalpy is about 20 kJ/mol G RT ) G cat = G N - G E Transition state binding by proline racemase Transition state analogs are strong inhibitors ...
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This note was uploaded on 03/26/2012 for the course CHEM 350 taught by Professor Lee during the Spring '08 term at SIU Carbondale.

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