Chapter 7: Enzyme Mechanisms Flashcards
What are enzymes and what do they do?
- Often proteins
- Enhance reaction rate
What is an enzyme made of RNA called
Ribozyme
What are the two models of enzymes and how do they differ?
Lock and key:
- Substrate binds to enzyme perfectly
- This means that the substrate will be unable to be released because of a tight fit
Induced fit:
- Enzyme is flexible
- Substrate will change conformation during catalysis
- Allows for larger number of weaker interactions between substrates and enzymes
What happens when a substrate binds to an enzyme in the induced fit model (Hexokinase example)
- Glucose binds to active site
- Conformational change blocking water from active site
- Phosphorylation is promoted
Enzymes usually bind to substrates with ____________ and ________. They also promote __________ reactions.
High Affinity
Specificity
Catalytic
Substrates binding to the active site induces _________ in the enzyme.
Changes
How is enzyme activity regulated in cells?
- Bioavalibility
- Catalytic efficiency
- Activity enzymes regulate covalent modification
How do enzymes affect the reaction rate, ratio of substrate to product, and ΔG?
- Speed up reaction rate
- Ratio of substrates and products remains at equilibrium (undisturbed)
- No Change in ΔG
How do enzymes speed up the rate of reaction?
- Decrease Activation energy
- Change ΔG‡
- Decrease of ΔG‡ by 5.7 kJ/mol results in 10-fold increase in reaction rate
How can the reaction 2H2O2 ⇋ 2H2O + O2 be sped up?
- Reaction normally takes 1 year for one mol
- Adding Fe2+/Fe3+ speeds time up to 12 minutes
- Adding catalase speeds up to 10^-7 seconds (10^15 rate increase)
How can X be transferred from A to B, what factors influence the reaction?
- Orientation must be right
- Energy must be sufficient enough for X to be transferred
How do enzymes influence the activation energy and ΔG‡ of a reaction?
- Decrease activation energy
- Decrease ΔG‡ resulting in increase of reaction rate
What are cofactors and what do they do?
- Small molecules that aid in catalytic reaction within active site
- Ex. Fe2+, Cu2+, Mg2+
- Help in catalysis when amino acid side chains are insufficient
What are coenzymes and what do they do?
- Enzyme cofactors that require organic component
- Include vitamin derivative species such as NAD+ and FAD+
What are prosthetic groups?
Coenzymes that are permanently associated with enzymes
Fill in the blanks with the following word bank: Cofactors, Coenzymes, Cosubstrates, Metal ions, Prosthetic groups
What is NADH and what does it do?
- Nicotinamide adenine dinucleotide (NAD+/NADH)
- Common coenzyme in oxidation/reduction reactions
- Carrier of electrons
What is Lipoamide and what does it do?
- Temporary carrier of acetyl group in reaction catalyzed by pyruvate dehydrogenase
- Permanently attached to enzyme
- Pyruvate + CoA + NAD+ → acetyl-CoA + CO2 + NADH
What two things are included in most enzyme names?
- Most enzymes end in -ase
- Substrate usually included in name
How does an enzyme’s structure increase the reaction rate?
- Lower activation energy by stabilizing transition state
- Provide alternate path for product formation
- Reduce entropy by orienting the substrates appropriately for reaction to occur
How do enzymes create products?
- Enzymes bring substrates together
- Geometric and chemical complimentarity
- High local concentration
- Optimal orientation
- Bind via non-covalent interactions
How do active sites contribute to catalytic properties?
- Sequester microenvironment of active site
- Provide optimal orientation and exclude excess solvents such as H2O
- Binding between substrate and enzyme create a transition state
- Presence of catalytic function groups
How does orientation influence the formation of Fructose 1,6 bisphosphate? (list steps)
- Dihydroxyacetone phosphate forms intermediate with Lys229 in enzyme with active site
- Glyceraldehyde-3-phosphate binds to active site
- Adol addition takes place in the active site forming 1,6 bisphosphate
- Because of the orientation when the substrates bind to the active site the formation of a product is possible.
How is water excluded in the phosphorylation of glucose in hexokinase? Fatty acid Isomerase?
- Water is excluded via conformational change
- This way phosphate is transferred to glucose instead of H2O
- Fatty acid isomerase creates a hydrophobic channel
What facilitates transition states?
Binding energy such as Van der Waals interactions, Ionic interactions, and H-bonds
What are powerful inhibitors of enzymes?
- Transition state Analogs
- Stable molecules that mimic the transition state
- Bind tightly to active site
How can you determine if an amino acid is in its acid or base form?
- Acid will be protenated or have a (+) charge
- Base will be deprotenated or have a (-) charge
How do amino acids participate in acid-base catalysis?
Function groups can act as acids or bases
How does covalent catalysis occur?
- Nucleophile group on enzyme attacks electrophile center on substrate making a covalent enzyme-substrate intermediate
How does metal ion catalysis occur?
- Metals used to promote proper orentation of bound substrates to aid in redox reactions
What is a coenzyme-dependent redox reaction?
- Energy conversion redox reaction such as CAC, ETC, or photosynthesis
- Include dehydrogenases
- Involve NAD+/NADH, NADP+/NADPH, FAD+/FADH2, and FMN/FMNH2
What is a Metabolic transformation reaction?
- Involve isomerizations, condensations, and dehydrations (hydrolysis) reactions
The transformation from 2-phosphoglycerate to phosphoenolpyruvate and the splitting of a polypeptide chair with chymotrypsin require _______
H2O
What is reversible covalent modification?
- Act as molecular switches that turn on and off cell signaling and gene expression
- Include kinases and phosphotases
- ATP is commonly used as a phosphoryl group source
What does chymotripsin do?
Hydrolyze peptide bonds
What do proteases do?
Cleave peptide bonds
- Peptide + H2O ⇋ Peptide 1 (COO-) + Peptide 2 (NH3+)
Why is a peptide bond more stable (than cleaved peptides)?
- Resonance structure provides partial double bond character
- Carbonyl carbon is less electrophilic and resistance to hydrolysis
What is the role of proteases in the stomach?
- pH 1-2
- kills bacteria
- unfold proteins
- pepsin
What is role of proteases in the pancreas?
- Bicarbonate secretion bring pH back to 7
- trypsinogen - converted to trypsin then breaks down proteins
-chymotripsinogen - precursor of chymotripsin which breaks down proteins into smaller peptides
-procarboxypeptidases - enzyme that hydrolyzes peptide bond at C-terminal
What is the role of proteases in the small intestine?
Zymogens convert to trypsin, chymotripsin, and carboxypeptidase
Where are zymogens produced?
The pancreas
What is a zymogen?
Precursor of an enzyme such as trypsinogen, chymotripsin,proelastase,procarboxy-peptidase, and prolipase
Describe the structure of chymotripsin.
- 245 amino acids
- Synthesized as chymotripsinogen (single chain)
- Activated by removal of 4 aminoacids at the boundaries of the chains
- 3 chains
- Chains held by disulfide bond
- Three critical residues for catalysis: His 57, Asp 102, Ser 195
How does chymotripsin catalysis work?
- General acid-base catalysis
- proton transfer by groups other than water
- Covalent catalysis
- Formation of powerful nucleophile
- Rate of hydrolysis increase by 10^9
Why is chymotripsin considered a serine protease?
It makes Serine a highly reactive nucleophile which is used to cleave proteins
What is the catalytic triad and what do they do?
- His 57, Asp 102, Ser 195
- Asp oreints His residue
- His positions and polarizes -OH of Ser
- His acts as a base catalyst (H+ acceptor)
- Ser wants to deprotenate
What is step 1 and 2 of chymotripsin cleaving?
- Polypeptide binds to active site
- His57 removes proton form Ser195 allowing nucleophilic attack by Ser O- on carbonyl carbon of the peptide
What is step 3 of chymotripsin cleaving?
- His57 donates a proton to amino group of substrate resulting in peptide bond cleavage
- carbonyl-terminal fragment released as first product
What is step 4 of chymotripsin cleaving?
- H2O (reactive) enters active site and His57 acts as base and deprotenates water, resulting -OH acts as nucleophile and attacks carbonyl carbon of covalent acyl-enzyme intemediate
What is step 5 and 6 of chymotripsin cleaving?
- His 57 donates proton to Ser 195 causing cleavage of acyl-enzyme resulting in the release of amino-terminal fragment
- Product 2 is released and catalytic triad is regenerated
Outline the major points in the catalytic mechanism for chymotripsin
- 2 nucleophilic attacks
- Ser and H2O attack carbonyl group
- Peptide bond cleavage
- Carboxy-terminal fragment released as 1st product
- N terminal fragment released
- Protein cleaves into 2 fragments
What are two other examples of enzymes with the same catalytic strategy (binding pocket)?
Chymotripsin, Trypsin, Elastase
What do statins do?
Inhibit cholesterol sythesis
What is the reaction rate of a first order reaction?
v = k [S]
k = rate constant
First order: one substrate and one product
What is the reaction rate of a second order reaction?
k = kBT/h e^(-ΔG‡/RT)
kB = Bolzman constant
h = Planck’s constant
How are enzyme kinetics measured?
Rate of substrate disappearance/ product formation as a function of time = velocity of reaction
How does [S] affect reaction rate in Michaelis-Menten Kinetics?
v0(initial velocity) = Reaction rate
v0 varies with [S]
What happens when [S] reaches a high concentration?
Eventually if the [S] is high enough vmax will be reached and enzyme activity plateaus
What is the equation for finding v0?
What is the Michaelis-Menten Constant?
What does v0 equal when in Steady-State conditions?
______ varies with [E]
Km is ______________ [E]
- Vmax
- independent of
What does the Lineweaver-Burk equation do
Plot Km/Vmax to get quantitative information
What is the Lineweaver-Burk Equation?
What is the v0 equation accounting for catalytic efficiency (Kcat)?
How efficient is enzyme activity at varrying pH for pepsin, Glucose-6-phosphotase, and Arginase?
- Pepsin 1.6
- Glucose-6-Phosphatase 7.8
- Arginase 9.7
How is DNA poly efficiency effected by temperature when using E. coli vs Taq?
- E. coli 37C
- Taq 80C
What two factors mediate enzyme activity?
- Bioavailibility (amount of enzyme in tissues)
- Control of Catalytic efficiency through
- Proteolytic modification
- Covalent modification
- binding of regulatory molecules
Compare reversible and irreversible enzyme inhibition
Reversible
- Noncovalent binding of small biomolecule/protein to enzyme subunit
- Competitive
- Uncompetitive
- Mixed
Irreversible
- Inhibitory molecule forms covalent or strong noncovalent bond with catalytic group in enzyme active site
- Kills enzyme through tight binding
- enzyme will never function again
What type of inhibitor is DFP (Diisopropylfluorophosphate) and what does it do?
- Irreversibly enzyme inhibitor
- DFP blocks protease and phospholipase enzymes
- Forms covalent bond with (specific) reactive serine in active site
- Kills enzyme
What type of inhibitor is Malonate and how does it work?
- Competes with succinate to bind to active site
- Competition often in cells
Diagram competitive inhibition, uncompetitive inhibition, and mixed inhibition
How can competitive inhibition be overcome?
Increase [S]
What type of inhibitor is Saquinavir and what does it do?
- An HIV protease inhibitor (competitive)
- Mimics Phe-Pro substrate
- Binds tightly to active site
What type of inhibitor is methanol and what does it do?
- Methanol(competitive) binds to dehydrogenase in the liver and is converted to formaldehyde
- Extremely harmful to tissue can result in blindness and death
- Therapy involves slow intravenous infusion of ethanol as a competitor to methanol resulting in the filtering out of methanol
How does uncompetitive inhibition work?
- Inhibitor binds to enzyme-substrate complex resulting in a conformation change of the active site
How does uncompetitive inhibition affect Vmax and Km-app?
- Decrease in Vmax and Km-app
How does competitive inhibition affect Vmax and Km-app
Vmax will be unchanged but Km-app will change
How does mixed inhibition work?
- Binding to site different to active site
- Can be a mix of competitive and uncompetitive inhibitors
How does mixed inhibition affect Vmax and Km?
- Decrease Vmax
- Increase or decrease in Km
Why are metabolic pathways controlled by a regulated enzyme?
- To maximize the efficiency of metabolic intermediates
What is feedback inhibition?
- End product of a pathway functions as an inhibitor in a prior enzyme in the pathway
Example: ATCase (aspartate transcarbamoylase)
What type of curve do regulatory enzymes exhibit?
- Sigmoidal curve (allostery)
- Sigmoidal is s shaped
How does ATCase work?
- Feedback inhibition
- CTP and ATP are allosteric regulators
Diagram and outline the steps in Allosteric regulation
Describe the ACTase organization
- C6R6
- 2 trimers C3R3
- R = regulatory subunit
- C = catalytic subunit
