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BIOC12H3 (56)
Lecture 10


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Biological Sciences
Shelley A.Brunt

lec10 outline 1. finish enzymes 2. coenzymes and vitamins activation of some pancreatic zymogens 1. what is a zymogen? a. inactive enzyme precursor 2. enzymes made in pancreas and then they get moved and cut into active form 3. trypsin a. cleaves various zymogens to activate them 4. feedback regulation not shown, but very much present comparison of polypeptide backbones (chymotrypsin / trypsin / elastase) 1. shows similar structure  common ancestor a. homologous two lobed enzyme with active site between the two domains i. substrate specificities are due to small structural differences in the enzymes 2. prof notes a. main players i. chymotrypsin ii. trypsin iii. elastase b. looks very similar, but are different enzymes i. differ in the depth and width of their active site 1. what has to fit into the active sites c. active sites contain these amino acids in the catalytic center i. serine ii. histidine iii. aspartate comparison of serine proteases 1. for determining enzyme composition / sequence a. trypsin i. catalyzes the hydrolysis of peptide bonds on the carbonyl side of arginine or lysine b. chymotrypsin i. less specific cleavage on the carbonyl side of uncharged residues with aromatic or bulky hydrophobic side chains 1. such as phenylalanine or tyrosine c. both trypsin and chymotrypsin contain a binding pocket that correctly positions the substrate for nucleophilic attack by an active site serine residue 2. each enzyme has an extended region in which the substrate polypeptides fit a. however, the specificity pocket near the active site serine is very different for each enzyme i. making it substrate specific 3. prof notes a. ability to sequence amino acids and cleave amino acids into peptides differ for each enzyme i. trypsin 1. catalyzes peptide bonds ii. chymotrypsin 1. less specific peptide cleavage on carbonyl side iii. key point 1. both cleaves amino acid on C-terminal site b. catalytic binding pocket will be slightly different towards trypsin and chymotrypsin i. serine is active amino acid in these enzymes (serine proteases) 1. serine is key component a. the specificity pocket near the active site serine is very different for each enzyme i. emphasis on this / what does this mean? binding sites in the serine proteases 1. different substrate specificities a. S1 pocket i. chymotrypsin 1. tyrosine (substrate) 2. hydrophobic pocket with uncharged serine at the base ii. trypsin 1. arginine (substrate) 2. aspartate is negatively charged a. forming an ion pair with arginine or lysine iii. elastase 1. alanine (substrate) 2. elastin is a fibrous protein rich in glycine and alanine 3. side chains of valine and threonine create shallow binding pocket that closes off pocket a. therefore binds amino acids with small side chains (alanine / glycine) 2. prof notes a. chymotrypsin i. slightly wider to accommodate aromatic ring of tyrosine b. trypsin i. deeper pocket 1. length of the arginine side chain c. elastase i. more shallow pocket to accommodate shorter side chains serine proteases 1. use both chemical and binding modes to control catalysis a. difference between chemical and binding modes? i. chemical mode ii. binding mode 2. e.g. chymotrypsin a. look at role of three catalytic residues (catalytic triad) i. his-57 ii. asp-102 iii. ser-195 b. mechanism can be applied to other hydrolases i. i.e. cleavage of amide or ester bonds via a similar process ii. what process? what mechanism? 1. similar mechanism to the superoxide dismutase mechanisms discussed in lec09 3. prof notes a. chymotrypsin role of 3 catalytic residues known as the catalytic triad i. his-57 ii. asp-102 iii. ser-195 b. mechanism applied to other hydrolases i. cleavage of amide or ester bonds via a similar process 1. what does this mean? catalytic site of chymotrypsin 1. catalytic triad a. active site residues are arrayed in a h bonded network i. the conformation of the three residues stabilized by a h bond between 1. the carbonyl oxygen of the carboxylate side chain of asp-102 and 2. the peptide bond nitrogen of his-57 3. no mention of serine? b. the triad is buried in a hydrophobic environment 2. prof notes a. catalytic triad i. histidine ii. serine 1. OH group 2. ability to donate protons iii. aspartate b. catalytic residues are arrayed in a h bonded network i. stabilizes active site to allow substrates to come into active site specificity pocket for chymotrypsin lined with hydrophobic residues 1. hydrophobic environment allows for phenylalanine in binding pocket catalytic triad of chymotrypsin 2. reaction cycle begins when his-57 extracts a proton from ser-195 a. ser-195 becomes a strong nucleophile that will attack the peptide bond i. what peptide bond? b. the reaction is favored because asp-102 stabilizes the histidine i. the histidine promotes the attack on serine 3. important a. normally the side chain of serine is not sufficiently acidic to undergo a deprotonation in order to serve as a strong nucleophile i. usually the hydroxylmethyl group has a high pKa 1. how does it achieve the above ionization? a. see prof notes 4. Prof notes a. Catalytic triad begins when histidine-57 extracts a proton from serine-195 b. What happens to serine-195 i. Becomes a very strong nucleophile that will attack the peptide bond c. Highly stable because aspartate-102 stabilizes histidine i. Histidine promotes attack on serine d. Serine potentially present in active site i. But not very common place 1. Normally not sufficiently acidic to undergo deprotonation a. However, the microenvironment allows this ionization i. Mechanism to do this is covalent and acid-base catalysis Mechanism of action 1. Includes covalent catalysis a. By a nucleophilic oxygen 2. General acid-base catalysis a. Donation of a proton to form a leaving group 3. Binding of a peptide (substrate) causes a small conformational change in chymotrypsin a. Which sterically compresses asp-102 and his-57 4. Low barrier h bond forms between asp-102 and his-57 side chains a. His-57 pKa rises from 7 to 11 i. This very strong bond drives electrons toward the second N atom of the imidazole ring of his-57 1. Making it more basic a. The increase in basicity makes his-57 an effective general base for removing a proton from the –OCH OH2of ser-195 i. This mechanism explains how the normally unreactive alcohol group of serine becomes a proton nucleophile 5. Prof notes a. Covalent catalysis i. Nucleophilic exchange b. Acid base catalysis (general) i. Donation of proton to form a leaving group c. Binding of peptide i. Leads to small conformational change in chymotrypsin 1. Sterically compressed asp-102 and his-57 leads to change in conformation a. During process that substrate alters binding site d. Causes low barrier h bond between asp-102 and his-57 side chains i. Change in conformation causes pKa rise from his-57 1. Created scenario where histidine is made into an effective general base e. Normally unreactive alcohol (serine) becomes a nucleophile Mechanism of chymotrypsin 1. Use text associated with a. figure 11 – figure 29 in Pratt b. figure 6.27 in Horton c. figure 9.7 in Berg d. figure 14.2 in Garrett 2. noncovalent ES complex forms a. substrate is oriented i. the binding interaction places the carbonyl carbon of the susceptible peptide bond (scissile bonds) next to the ser-195 1. R1 binds to hydrophobic pocket a. Compression of asp and his i. The strain is relieved by formation of low barrier h bond between asp and his 1. Allowing removal of proton from ser 3. The nucleophilic oxygen of ser-195 attacks the carbonyl carbon of the target peptide bond a. Forms a tetrahedral intermediate (E-Ti ) 1hich resembles the transition state i. Transition state (four atoms bonded to a carbon) 4. Once Ti f1rms a. The substrate CO bond changes from a double bond to a longer single bond b. This allows the negatively charged oxygen of Ti to move to a position called the 1 oxyanion hole i. Oxyanion hole with gly-193 and ser-195 1. This oxyanion hole stabilizes the tetrahedral intermediate 5. The imidazolium ring of his-57 acts as an acid base catalyst a. Donating a proton to the nitrogen of the scissile peptide bond i. Result is cleavage of amine product 6. The carbonyl group from the peptide forms a covalent bond with the enzyme a. Forming an acyl-enzyme intermediate i. The product with a new amino terminus leaves the active site 7. Water enters the active site a. Replacing the amine product b. It is held in place by his-57 by proximity effect i. His-57 removes a proton from water to provide an OH- group to attack the carbonyl group of the ester 1. Result is hydrolysis (deacylation) of the acyl-enzyme intermediate 8. A second tetrahedral intermediate called E-Ti is2formed and stabilized by an oxyanion hole 9. His-57 is now an imidazolium ion again a. Donates a proton resulting in the collapse of the second intermediate 10. The second product is formed a. A protein with a new carboxy terminus 11. The carboxylate product is released from the active site a. The enzyme is regenerated 12. Prof notes a. Mechanism by which it happens i. Target amino acid that enters the active site 1. Important glycine sitting at the binding site ii. In order to reduce strain 1. Low barrier h bond between aspartate and histidine a. Allows removal of proton from serine iii. Carbonyl carbon of amino acid of susceptible peptide bond (scissile bond) is next to ser-195 1. Scenario where nucleophilic oxygen on serine-195 attacks the carbonyl carbon of the target peptide bond iv. Stabilization by oxyanion hole 1. What is the oxyanion hole? 2. Formation of this hole is about a. Stabilization of the enzyme b. Ability to form stable intermediate i. Important for it to carry out its function v. Histidine now acts in acid base catalysis vi. Cleavage of amine product 1. Carbonyl group still attached a. Formation of acyl-enzyme intermediate vii. Water comes in 1. Held in place by histidine due to proximity effect a. Result in hydrolysis (acylation) of the acyl enzyme intermediate i. Role for 1. Water 2. Hydrolysis 3. … 2. Missed some points of slide 14 / 15 / 16 viii. Very complex process 1. Need to know what the various intermediates are a. Therefore need to know the various steps i. Cannot memorize, must understand Stabilization of the intermediates at the oxyanion hole via hydrogen bonds 1. Transition state stabilization a. Carbonyl carbon of scissile peptide is constrained from binding to the oxyanion hole b. Tetrahedral intermediate i. With the charged carbonyl oxygen of the scissile peptide (oxyanion) enters the oxyanion hole 1. Forms h bonds to backbone NH of gly-193 and ser-195 a. Conformational distortion i. Results in binding of intermediate 2. Prof notes a. Stabilization that is important i. Binding of catalytic triad stabilized by gly-193 1. Holding substrate in correct orientation to allow the tetrahedral intermediate to form a. Causes conformational distortion i. Allows enzyme to function properly Review: serine proteases use many catalytic modes 1. Steps 1-4 a. Forward reaction used proximity effect i. i.e. gathering of reaction (substrate and enzyme) 2. Acid base catalysis by histidine in steps 2 and 4 lower the energy barrier 3. Covalent catalysis using the –CH OH of serine occurs in steps 2-5 2 4. The unstable tetrahedral intermediates at step 2 and 4 are similar to the transition states for these steps 5. H bonds in the oxyanion hole stabilize these intermediates 6. Serine proteases use both chemical modes (acid base and covalent catalysis) and binding modes (proximity effect and transition state stabilization) a. Chemical modes i. Acid base catalysis ii. Covalent catalysis b. Binding modes i. Proximity effect ii. Transition state stabilization 7. Prof notes a. Proximity effect i. Gathering event 1. Brings enzyme and substrate into close proximity b. Mostly read off the slide c. How well you can stabilize unstable intermediate i. Results in how well enzyme will function 1. Unstable intermediates have more in common with transition states than stable intermediates d. Missed a point emphasizing the last point(s) before change of slide Site directed mutagenesis 1. Used to show significance of ser-his-asp catalytic triad a. In the bacterial serine protease subtilisin i. Replacing 1. The covalent catalyst serine or 2. The acid base catalyst histidine a. With alanine reduced enzymatic activity significantly ii. But mutation of all three amino acids to nonconserved amino acids 1. The enzymatic rate was still well above the nonenzymatic rate due to the role of transition state stabilization 2. Prof notes a. Missed this slide  did not hear much of the beginning b. If we mutate them into a different amino acid i. Results in change in enzymatic activity (rate wise) ii. Even when you mutate all three triad amino acid 1. The enzymatic rate was still well above non-enzymatic rate a. Because of the transition state stabilization i. Therefore transition state stabilization is crucial in the catalysis between enzyme and substrate to product 2. Knocking out all three catalytic amino acid residues results in activity very similar to knocking out only one of them Carboxypeptidase II from wheat 1. Has a catalytic triad composed of the same amino acids of chymotrypsin a. But it has no structural similarity i. Suggests that the his-ser-asp triad is important in the catalytic site 2. Prof notes a. No structural similarity between carboxypeptidase II with chymotrypsin i. But has a catalytic triad composed of the same amino acids of chymotrypsin 1. Important triad in catalysis Other major peptide cleaving enzymes 1. Major peptide cleaving enzymes a. Cysteine b. Aspartyl c. Metalloproteases 2. These are three alternate approaches to peptide bond hydrolysis a. i.e. not based on active serine residues b. Generate a nucleophile that attacks the peptide carbonyl group i. instead of serine, these major peptide cleaving enzymes facilitate the mechanism from substrate to product 3. Prof notes a. Use of general nucleophile that attacks a carbonyl group b. When you want to inhibit proteases i. You have to use cocktail of protease inhibitors 1. Because various inhibitors are needed when different proteases have different active sites Cysteine proteases 1. Cysteine residue activated by histidine a. Carries out a nucleophilic attack on the peptide 2. Prof notes a. Papain has two important players i. Cysteine 1. If it is a cysteine protease, of course active player is cysteine ii. Histidine Aspartyl proteases 1. Pair of aspartic residues act together in conjunction with water to attack the peptide bond a. E.g. HIV-1 / proteases / pepsin 2. Prof notes a. Important players i. Aspartate b. Common in HIV-1 / proteases / pepsin i. One of the things to put into HIV is a protease inhibitor cocktail 1. Inhibits ability of virus to infect other cells Metalloproteases 1. Active site has metal ion a. The metal ion is almost always zinc i. Zinc activates water to carry out a nucleophilic attack on peptide 2. Prof notes a. Requires metals to carry out cleavage i. Active site requires metal 1. E.g. zinc that activates water to carry out a nucleophilic attack on peptide Cofactors and co-enzymes 1. Often ignore cofactors and coenzymes because they complicate things 2. Many enzymes need cofactors for catalysis 3. These cofactors are often required to convert inactive enzymes to active enzymes a. Inactive enzymes are called apoenzymes b. Active enzymes are called holoenzymes 4. Cofactors include a. Essential ions i. i.e. metal b. Organic compounds i. Called coenzymes 5. Prof notes a. Most enzymes require some sort of co-factor i. Whether co-substrate or co-enzyme b. Cosubstrate i. Substrates in reaction breaks down c. Coenzyme i. Enzymes in reaction regenerates d. Required to convert apoenzymes to holoenzymes Cofactors 1. Cofactors made up of a. Essential ions i. Activator ions 1. Loosely bound 2. Activator ions are reversibly bound and often participate in binding substrates ii. Metal ions of metalloenzymes 1. Tightly bound 2. Some cations are tightly bound and participate directly in catalytic reactions b. Coenzymes i. Cosubstrates 1. Loosely bound ii. Prosthetic groups 1. Tightly bound 2. Specific for the chemical groups that they accept and donate a. For some hydrogen or electron b. For others large covalently attached groups 3. Attached at reactive center 4. Can be derived from vitamins 2. Prof notes a. Coenzymes can be thought of as group transfer reactions b. Cosubstrates i. Altered in reaction ii. Very different from enzyme iii. Broken down in reaction c. Prosthetic groups i. Stays the same ii. Regenerated iii. AKA coenzymes iv. About accepting and donating chemical groups v. Can be derived from vitamins Essential ion cofactors 1. More than 25% of known enzymes require metallic cations for full catalytic function a. Two groups i. Metal activated enzymes 1. Have an absolute requirement for added metal ions 2. Alternatively stimulated by the addition of metal ions 3. Monovalent cations such as K+ or divalent such as Ca2+ or Mg2+ a. E.g. kinases require Mg2+ for the magnesium-ATP complex used for the phosphoryl group donating substrate i. The magnesium shields negatively charged phosphate groups of ATP 1. Making them more susceptible to nucleophilic attack ii. Metalloenzymes 1. Contain tightly bound metal ions at their active sites 2. Usually transition metals
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