Biochem Chapter 6 Flashcards
Who discovered enzymes?
Edward Buchner
How do enzymes differ from ordinary chemical catalysts (4 ways)
- Can accelerate biochemical reactions faster
- Catalyze under mild conditions
- Give 100% yield
- Can be regulated
What kinds of bonds can an enzyme form with a substrate?
- Hydrophobic Effect
- Hydrogen Bond
- Ionic bond
- Covalent bond
Who developed the Lock and Key model of enzyme-substrate binding?
Emil Fischer
What are limitations of the lock and key model?
- Does not account for enzyme’s ability to bind diverse substrates
- Does not account for enzymes stabilizing transition state of rxn
Who developed the induced fit model?
Koshland
What is a cofactor?
Non-protein component
What is an apoenzyme?
Protein component (without cofactor)
What is a haloenzyme?
Apoenzyme + cofactor
What is a prosthetic group?
When the cofactor is tightly bound to the protein component
What 5 factors affect enzyme activity?
- Temp.
- pH
- Cofactors
- Allosteric Effectors
- Inhibitors
What are the 6 catalytic mechanisms for enzymes?
- Proximity Effects
- GABC
- Covalent (nucleophilic) catalysis
- Electrostatic stabilization
- Preferential stabilization of strained transition state
- metal ion catalysis
Describe Proximity effects
Positioning molecules close together for rxn
When you bring substrates close together for proximity effects, how is the significantly unfavorable loss in entropy paid for?
By the very favorable deltaH of enzyme + substrate binding
Describe GABC
Donating/Accepting a H+ by providing a weak acid/base group to stabilize an unstable TS & thus lower Ea
What groups can participate in GABC?
Tyr, Cys, Lys, Arg, His, Asp, Glu
Describe the roles of His 119 and His 12 in the RNase A Mechanism
Step 1: His 12 acts as a general base, abstracting a proton from 2’ hydroxyl of 3’ nucleotide ……. His 119 acts as a general acid, donating a proton to 5’ hydroxyl of nucleoside
Step 2: His 12 acts as a general acid, donating H+ to make ROH a good LG ……… His 119 acts as a general base, deprotonating H2O to make it a better nucleophile
What are the pKas of His 119 and His 12
His 119 = 6.4
His 12 = 5.4
Describe Covalent (nucleophilic catalysis)
Enzyme active site provides a nucleophile that forms a covalent Enz-S adduct that ALTERS THE REACTION PATHWAY (& thus lowers Ea)
What groups can undergo Covalent (nucleophilic) catalysis
Tyr, Cys, Lys, His, Asp, Glu, Ser, Thr
What is Lysine commonly used in the formation of?
Schiff bases/imines
What is the selectivity of Trypsin, Chymotrypsin, Elastase
Trypsin: Lys, Arg
Chymotrypsin: F, T, W
Elastase: Gly, Ala, Val, Ser
What catalytic mechanisms are involved the serine protease mechanism?
- GABC
- Covalent Catalysis
- Electrostatic Stabilization
Describe Electrostatic Stabilization
Charge distribution in active site that electrostatically stabilizes an unstable charged TS & thus lowers Ea
What are the roles of Asp 102, His 57, and Ser 195 in the Serine Protease Mech?
Asp 102: Electrostatic Stabilization (whole mech.)
Ser 195: Covalent Catalysis
His 57: Starts out as a general base
Describe the two factors of electrostatic stabilization in the serine protease mechanism
- Tetrahedral Intermediate steps (2 TS of rxn) have unstable oxyanions that fit perfectly into the OXYANION HOLE
- In both steps, deltaH+’s of backbone amide NH’s provide opposite charge to electrostatically stabilize oxyanion
What is TPCK? What is it important in the elucidation of the serine protease mechanism?
TPCK is a reactive substrate analog that binds to His 57, proving that it is important for catalysis in the serine protease mechanism
What is the difference between TPCK and TLCK?
TPCK: Reacts with His 57 of Chymotrypsin
TLCK: Reacts with His 57 of Trypsin
What is DIPF? Why is it important?
DIPF irreversibly binds to Ser 195 of serine protease mechanism, proving that Ser 195 is important for the reaction
What amino acids are involved in the catalytic triad?
Asp 102, His 57, Ser 195
What is a Zymogen? Why are they important? How are they made active?
Enzymes that are synthesized with an extra stretch of protein that blocks active site and prevents substrate binding ……… Proteins are made in pancreas, so they need to be inactive until they get into the lumen of the duodenum ……… Proteolysis cuts off piece of protein blocking active site
Describe the “snowball effect” that has to do with Trypsin
Protease activates Trypsin, then Trypsin can activate itself
What is pancreatic trypsin inhibitor?
Binds trypsin very tightly to prevent trypsin digestion of the pancreas
What are the characteristics of the binding pockets for Trypsin, Chymotrypsin, and Elastase?
Trypsin: Deep, (-) charge
Chymotrypsin: Deep, hydrophobic, able to accept rings
Elastase: Shallow, uncharged
Is Thrombin a member of the serine protease family? What is Thrombin significant for?
Yes; Thrombin is important in clotting cascade (thrombin, fibrinogen, fibrin)
Preferential Stabilization of Strained Transition State
Lowering Ea by stabilizing a conformationally unstable TS
What is the role of lysozyme in the body?
Destroys polysaccharide chains of bacterial cell walls
Which bond does Lysozyme break?
Hydrolyzes the Beta glycosidic bond between D-NAM and E-NAG
What is unique about D-NAM sugar in the lysozyme mechanism?
D-NAM in active site is in half chair conformation
How many carbohydrate residues can lysozyme bind in its active site?
6 carbohydrates
What residues are important in Lysozyme’s active site?
Asp 52 and Glu 35
What enzymatic mechanisms does Lysozyme utilize?
- GABC
- Covalent Catalysis
- Preferential Stabilization of strained TS
What are the roles of Glu 35 and Asp 52 in the Lysozyme mechansims?
- Glu 35 - GABC (starts as general acid)
- Asp 52 - Covalent Catalysis
What is the difference between the old and new lysozyme mechanism?
In old mechanism, Asp 52 was believed to participate in electrostatic stabilization of carbocation intermediate, rather than covalent catalysis
What is a Transition State Analog?
Stable analogs that resemble transition state conformation of a rxn …. bind very tightly to the active site and inhibit enzyme activity
Describe Metal Ion Catalysis
Metal ions catalyzing rxns - by charge shielding (i.e. electrostatic stabilization involving metal ions); polarizing water to make it a better nucleophile, etc.
What are the 2 types of metal-containing enzymes? What is the difference between the two?
Metalloenzymes - Contain tightly bound metal ions
Metal-activated enzymes - loosely bind ions
What are the 4 ways that metal ions can aid in catalysis?
- Bind substrates to orient them for catalysis
- Through redox reactions
- Through charge stabilization (shielding)
- By ionizing/polarizing water
What kind of enzyme is carbonic anhydrase?
Metalloenzyme
What are the 6 IUBMB classifications of enzymes?
- Oxidoreductase
- Transferase
- Hydrolase
- Lyase
- Isomerase
- Ligase
Describe (1) Oxidoreductases
Enzymes that catalyze oxidoreduction reactions ….. the hydrogen acceptor is REDUCED
What cofactors participate in redox reactions?
NAD+, NADP+, FAD
Describe (2) Transferases
Transfer of a functional group from one molecule (donor) to another (acceptors)
Describe (3) Hydrolases
Cleave bonds by adding water
Describe (4) Lyase
Add water, ammonia, or CO2 across double bonds, or remove these elements to produce double bonds
Describe (5) Isomerases
They move groups around, giving you isomers
Describe (6) Ligases
Tie things together …. NEED ENERGY
What are catalytic antibodies?
“Artificial Enzymes”; Antibodies whose binding sites are complementary to the TS or a reaction (usually antibodies bind ground states)
