Ch11-120319 - CHEM 350: Introduction to Biological...

Info iconThis preview shows page 1. Sign up to view the full content.

View Full Document Right Arrow Icon
This is the end of the preview. Sign up to access the rest of the document.

Unformatted text preview: CHEM 350: Introduction to Biological Chemistry Brian Lee, Ph.D. brianlee@siu.edu Office: Neckers 146G or 324 Phone: 453-7186 Hours: 9:30am to 10:30am or by appointment Website: https:/ /online.siu.edu 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) (http://www.siu.edu/~fao/jobs/) Undergraduate Assistantships (http://undergraduateassistantship.siuc.edu/) McNair Scholars Program (http://www.siu.edu/~mcnair) REACH Awards Competition (http://www.siu.edu/~reach/) Assignments Read Chapter 12 Enzyme Kinetics Chapter 12 Problems Student Companion site for Voet, Voet & Pratt http://bcs.wiley.com/he-bcs/Books?action=index&bcsId=4274&itemId=0470129301 Third Midterm Exam, Wednesday March 28th Chapters 10-13 Help Desk Tuesday 6:30 to 7:30 pm in Neckers 218 Thursday 5:00 to 6:00 pm in Neckers 410 Catalytic Mechanisms • Acid-base catalysis • Covalent catalysis • Metal-ion catalysis • Electrostatic catalysis • Proximity and orientation • Transition state binding Lysozyme - antibacterial agent, lyses bacterial cell walls -attacks carbohydrates in peptidoglycan -cleaves glycosidic bond between Mur2Ac and GlcNAc -Mur2Ac: N-acetylmuramic acid (NAM) -GlcNAc: N-acetylglucosamine (NAG) found in tear drops an d egg whites cleaves between D and E Transition state binding to the distorted half-chair conformation of NAM promotes cleavage of the glycosidic bond. Isotope labeling to identify scissile bond Page 343 Mechanism of reaction without enzyme using general acid catalysis. Note, carbocation intermediate must be stabilized. Lysozyme uses: Glu35 COOH high pKa due to nonpolar environment Asp52 COO- stabilizes the carbocation-oxonium ion Glu35 COOH acid catalysis. Asp52 COOelectrostatic catalysis. Covalent Catalysis Asp52 COOparticipates in nucleophilic attack on the carbocation intermediate. Structural evidence of a covalent intermediate using fluorinated substrate analog. C2-F slows product release and C1-F helps covalent intermediate formation. NAM(B)-NAG(C)-NAM(D) noncovalent complex (yellow) D ring distorted half chair covalent complex (green) Serine Proteases Chymotrypsin - peptide bond cleavage on carboxyl-terminal side of large hydrophobic side chains: tryptophan, tyrosine, phenylalanine & methionine Covalent modification - nucleophilic catalysis Ser195 is highly reactive Chymotrypsin serine protease cleaves after Trp, Phe or Tyr (also Leu or Met) Catalytic triad: Ser, His and Asp psin lysine specificity Evidence for an acyl-enzyme intermediate from the “burst phase” of pre-steady state kinetics. Chymotrypsin is capable of ester hydrolysis. a) p-nitrophenol is rapidly formed at nearly the same stoichiometry as the enzyme used in the reaction. b) Then a constant “steady-state phase” of release. Chymotrypsin follows a Ping Pong Bi Bi Mechanism E-OH + pNPhAc -> E-pNPhAc -> E-Ac + pNPh E-Ac + H2O -> E-Ac-H2O -> E-OH + Ac Enzyme activity is often dependent on pH. The optimal pH for catalytic activity usually matches the physiological conditions of the enzyme. pH 7.8 pH 1.6 His57 participates in catalysis Chymotrypsin rate dependence on pH kcat dependence on pH His57-imidazole pH 8.0 KM dependence on pH Ile16-Asp194 salt bridge affects substrate binding pocket Ile16-NH3+ Activation of Serine in the catalytic triad Catalytic triad: Asp102 -> His57 -> Ser195 Substrate binding: -hydrophobic pocket His57 acts as a general base catalyst and activates Ser195 hydroxyl to attack the carbonyl carbon. Asp102-His57 low barrier H-bond raises pKa of His57 For histidine to act as a base catalyst, it must be completely deprotonated or ... pKa 14.5 pKa His57 13 Ser195 pKa = 6.04 ... the first pKa must be much higher than 6.04. Problem: How do you remove a proton from Histidine (pKa 14.5) with an Aspartate (pKa 3.9) pKa 14.5 His57 pKa = 3.90 Asp102 pKa = 6.04 IMPOSSIBLE: This cannot be done in a normal acid-base reaction. Hydrogen bonding between histidine and aspartate will increase the pKa of histidine... Asp102 His57 pKa = ??? ...but not enough to abstract a proton from serine. Ser195 Asp102 His57 pKa = ??? If the hydrogen bond is stronger, then the pKa will increase enough to allow histidine to act as a base. What is a Low-Barrier Hydrogen Bond (LBHB)? Strong than normal. about 60 kJ/mol versus 20 kJ/mol If the LBHB forms in response to substrate binding, then the increase binding energy directly contributes to GB and the reduction in G . Note: Aps102 has a lower (or equal) pKa than His57, and normally would not be able to accept a proton from His57. The LBHB is the “best” explanation of how Asp102 helps to partially abstract a proton from His57 Oxyanion hole stabilizes negatively charged carbonyl oxygen in the tetrahedral intermediate through H-bonds. acyl-enzyme intermediate His57 can now act as a general acid catalyst, donating a proton to the new amino group. The carbonyl double bond reforms as the peptide bond is broken. First product is released in accordance with Ping Pong mechanism The acyl-enzyme intermediate is a long lived covalent complex. Waiting for water… Water binds in the same location as the departed amino group, stabilized by H-bonding to His57. Reversal of previous reactions to release second product and regenerate the enzyme. His57 now acts as a general base and removes a proton from water to create a nucleophilic hydroxide ion. The tetrahedral intermediate now involves the bound water molecule. Collapse of the short-lived intermediate His57 acts as a general acid to donate a proton back to Ser195. The second product is released and the enzyme has returned to it original state. Evidence for the tetrahedral intermediate: BPTI prevents trypsin from digesting the pancrease and binds to trypsin with 0.1 x 10-12 M affinity. X-ray structure of BPTI bound to trypsin shows the tetrahedral intermediate prior to breaking the peptide bond The structure of another serine protease, elastase, shows the second tetrahedral intermediate with water. pH 5.0 structure with water bound pH 9.0 structure of tetrahedral intermediate water Chymotrypsin (EC 3.4.21.1) Classification - 3) Hydrolase: hydrolysis reaction (transfer of functional groups to water) Catalytic Mechanisms -acid-base catalysis (His57) -covalent catalysis (Ser195) -electrostatic catalysis (oxyanion hole) -transition state binding More serine protease enzymes Trypsin is closely related to chymotrypsin The substrate binding pocket of trypsin has an Aspartic acid residue at the bottom of the pocket Specificity changes from large hydrophobic to large basic residues: Lys and Arg. Unrelated serine proteases have evolved independently and contain the same catalytic triad residues: Asp, His, Ser Other Classes of Proteases Cysteine protease papain caspase Aspartyl protease renin pepsin HIV protease Metalloprotease thermolysin carboxypeptidase A Zymogens – inactive precursors of enzymes. Digestive enzymes are first synthesized as zymogens to prevent digestion of tissue and organs. As trypsinogen leaves the pancrease, it is initially activated by enteropeptidase. Trypsin can also cleave trypsinogen for autocatalytic activation The fibrin network in blood clots is formed through the cleavage of fibrinogen by thrombin. Thrombin is activated by a cascade of clotting factors. Many of the clotting factors are proteases. ...
View Full Document

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.

Ask a homework question - tutors are online