Chapter 8 - Mechanisms and inhibitors
Terms in this set (21)
What are the 4 Catalytic Strategies used by enzymes?
1. Covalent Catalysis - active site contains a nucleophile (reactive gr) which is attracted to the pos charge (only briefly covalently modified
2. General Acid-Base Catalysis - molecule other than water accepts or donates proton
3. Metal Ion Catalysis - number of ways including serving as an electrophillic catalyst (attract e)
4. Catalysis by approximation and orientation - two substrates brought together - orientation facilitates catalysis
What is the effect of temp on enzymes?
Increase in temp can speed up a reaction - a decrease is a result of protein denaturation from max temp being surpassed
Increase in temp increases Brownian motion - makes interaction between enzyme and substrate more likely
What is the pH profile for a hypothetical enzyme?
pH dependence of enzyme due to presence of ionizable R groups (pH decreases -> [H+] increases)
How does Phsohofructose Kinase regulate pH?
-critical enzyme in glycolysis
-anaerobic exercise - Lactate + protons - increase pH in muscle
-activity of enzyme decreases as pH falls too much/fast reducing metabolism of glucose to lactic acid
-subunits of this enzyme to dissociate with idecrease of pH rendering the enzyme inactive and decrease lactic acid production
What is the difference between Reversible and Irreversible enzyme inhibitors?
Reversible - bind then dissociate from enzyme eg. Ibuprofen
Irreversible - permanently bond or dissociate very slowly eg. Aspirin
What are the types of reversible inhibition?
Competitive, Uncompetetive, Noncompetitive
Inhibitor mimics substrate
-competes for binding to active site
-effectiveness depends on affinities for site
-can be relieved with in increase in substrate concentration
-inhibitor binds to enzyme-substrate complex only
-site created only when enzyme binds substrate
-cannot be overcome by substrate concentration
-bind at site other than the active site but does not effect substrate binding (type of allosteric)
-acts by decreasing overall number of active enzyme molecules
Increase in competitive inhibitors increases [S]
-higher [S] required to attain reaction velocity
-high [S] can completely relieve inhibition
-has no effect on Vmax and increases Km
-inhibitor bnids only to enzyme-substrate complex therefore Vmax cannot be attained even at high [S]
-Vmax and Km reduced by equal amounts
-Km lowered as more inhibitor added - ES together longer results in increase affinity therefore lower Km
-binds to both free enzyme and ES complex
-Vmax cannot be attained
-Km unaltered therefore reaction rate increases more slowly at low [S]
-Vmax decreases - enzyme affect
What are the characteristics of Irreversible Inhibitors?
-target specific R groups of AA
-enzyme turns compound into inhibition
eg. penicillan acts by covalently modifying enzyme transpeptidase preventing the synthesis of bacterial cell walls therefore killing bacteria
-if treatment with irreversible inhibitor results in loss of enzyme activity - this loss suggests the modified functional groups are required for enzyme activity
What is Chymotrypsin?
-A serine protease (pancreatic - utilized to digest proteins - break peptide bonds in the middle of protein sequence)
-Hydrolysis - internal peptide binds next to bulky hydrophobic groups - uses 2 steps - involved covalently linked intermediate
Step 1 - fast, Step 2- slow
-The enzyme employs powerful nucleophile to attack nonreactive carbonyl gr of substrate which becomes covalently attached to substrate breifly for catalysis
What are the features of Step 1 and Step 2 of the Hydrolysis of Chymotrypsin?
Step 1 - acyl group of substrate becomes covalently to residue of enzyme as product being produced
Step 2 - The intermediate formed above is hydrolyzed to release carboxylic acid component of substrate and regenerate free enzyme
-takes longer for enzyme to be rest by hydroylsis of intermediate
What is the Catalytic Triad?
Polypeptide chain of enzyme - triad of AA residue in active centre that includes Serine, His, and Asp.
How does the Triad work together?
His - can get pronated at biological pH - gets proton from Serine and generates highly reactive Alkoxide ion
Serine - alkoxide ion attacks the substrate peptide bond
Asp - orients the histadine to accept proton from Serine
What are the steps of peptide hydrolysis?
1. Substrate binding
2. Serines nucleophillic attack on peptide carbonyl gr
3. Collapse pf the tetrehedral intermediate
4. Release of amine component
5. Water binding
6. Waters nucloeophillic attack on acyl-enzyme intermediate
7Collapse of the tetrehedral intermediate
8.Release of the carboxylic acid component
What is the oxyanion hole and how does it stabalize tetrahedral intermediates ?
-Oxyanion hole provides hydrogen bonding with the oxygen that shifts from going from C double bonded to O to C single bonded to O
-the hydrogen bonds link peptide NH groups and neg charged O atom of intermediate
What is the catalytic role of His?
have chrymotrypsin react with molecule that:
-specifically binds to active site bc resembles substrate
-then forms stable covalent bond with group on enzyme that is in proximity
What are the steps in the Triad?
Step 1 - reaction begins with O atom of side chain S195 making nucleophillic atack on carbonyl carbon atom of target peptide bond
Step 2 - Now 4 molecules bonded to carbon in a tetrehedron (intermediate) has neg charge on O atom from carbonyl group - oxyanion hole stabalizes the intermediate which contributes to binding E that helps stabalize transition state