BCHM-3050 Lecture Notes - Lecture 11: Metabolic Pathway, Protease, Deprotonation
8 Jun 2018
School
Department
Course
Professor
Enzymes and enzyme classes
Enzyme: used by living organisms to accelerate bio rxns
Bio and protein based
○
Lower activation energy of rxn
○
Neither consumed or reduced by rxn
○
•
Classes of enzymes
Oxidoreductases
Transferases
Hydrolase
Lysases
Isomerase
Ligase
Ex. Alcohol
dehydrogenase
Through
reduction/oxidation
Ex. Hexokinase
Take one part of
compound and put it
on the next
Ex. Cleavage of
peptide bond
using H2O
Has to be water
that cleaves the
compound using
hydrolase
Ex. Pyruvate
decarbonylase
Cleave single
compound into
two parts
Ex. Malaeate
Groups are
added.
Compound A is
isomerized to
compound B
Used to
glue to
compounds
together
Enzymes stabilize transition state
Activation energy: energy required by a starting specie to undergo rxn
•
Climb to transition state decreases from non catalyzed to catalyzed
Activation energy decrease from non catalyzed to catalyzed
○
•
Free energy of product and reactant = unchanged
Hess law and state function
○
•
Raising T increase the rate constant by increasing average kinetic energy
of the substrate and increases K
Temp = avg of kinetic energy
○
•
Lock and key model
Lock and key model: substrate fits into enzyme in specific manner
For every lock there is a specific key
○
Specific shape of substrate will fit to enzyme
○
•
Induced fit model: conformational changes in enzyme structure to fit
incoming substrate
Stabilize transition state through conformational change
Help promote rxn
§
○
Favored model
○
•
Enzymatic catalysis proceeds by one of 5 ways
Catalysis: rate enhancement of rxn
For each enzymatic rxn you can use one of the following:
General acid/base catalysis (GABC)
H bond network
□
Series of protonation and deprotonation
□
§
Covalent catalysis
Stabilize a particular transition state
□
§
Electrostatic stabilization
Bring in charged species or high EN specie to stabilize
the transition state
Want to neutralize
®
□
§
Proximity effects
Van der waal
□
§
Preferential stabilization of transition state
Via active site residue
□
Enzyme change shape
□
§
○
•
Protein conformation = role in catalysis
•
Enzymes must stabilize the transition state to achieve rate enhancement
Have different paths that yield the same results
○
•
Environmental effect on enzyme activity
Activity of enzyme depends on the number of local environmental
conditions
•
pH
Important individually
○
Change in pH can change the rate of protonation and deprotonation
○
•
Temperature
•
Ionic strength
Protein solubility
○
•
Substrate/enzyme concentration
•
Inhibitors /activators
•
Each enzyme has optimum set of conditions
Allow peak enzyme activity
○
•
Lysozyme
Cleave polysaccharide
Long chains of NAM and NAG
Cleave between 4th and 5th C on 6C chain
§
○
•
Has cleft
Clamps onto polysaccharide here
○
•
Catalytic mechanism (2 different ones):
Both start and end in the same place
○
Share half chair oxocarbenium ion intermediate
○
•
Philips mechanism
Oxocarbenium ion (+) stabilized by Glu/Asp residue (-)
○
•
Koshland mechanism
Form covalent adduct
No charge stabilization
§
○
•
Lysozyme activity v pH
Lysozyme works well in acidic environments
•
Glu35: must be protonated to act as general acid catalyst in rate
determining step
Want pH under pka of side chain
○
•
Asp52: deprotonated to attack oxonium ion
Has pKa of 3.86
○
pH > 3.6: deprotonate aspartate side chain
○
pH < 3.8: protonate side chain aspartate
○
•
Basically! Glu35 is protonated by Asp52. Asp52 is deprotonated and
makes a good nucleophile which attacks the oxonium ion
•
Protease
Chymotrypsin: serine protease
Enzyme that breaks peptide bonds
○
•
Catalytic triad
Conserved catalytic residue
○
•
Asp (D102) deprotonates His(H57) which then deprotonates Ser (S195).
Ser then nucleophilic attacks the carbonyl carbon on the incoming
polypeptide
•
Mechanism:
Oxyanion hole stabilizes and use residue to stabilize oxygen
○
•
Non protein molecules
Cofactors: non protein compounds
Utilize for enzyme activity
○
Role in enzyme function
○
•
Prosthetic group: non protein group that are tightly bound to enzyme
Not covalent
○
Ex. Heme group
○
Play structural role in enzyme function
○
Stay with enzyme instead of moving between enzyme
○
•
Two categories:
Inorganic (cofactors)
Metals
Common: Fe, Cu, Zn, Mn, Co
□
Rare: Ni, Mo, V, Se
□
Mg: not catalytic
Used for coordination and structure
®
□
§
○
Organic (coenzymes)
Vitamin derived compounds (ex. NADH or FADH)
§
Enzyme associated is transient
§
Cosubstrates when they travel b/w enzymes
§
○
•
Apoenzyme: enzymes w/o cofactor
•
Holoenzyme: with cofactor
•
Overview:
Enzyme
Holoenzyme (complex)
Protein and non protein
§
Apoenzyme: protein part
§
○
Simple: only protein
○
Cofactor
Prosthetic
Small inorganic molecule or atom
□
Tight bound apoenzyme
□
§
Coenzyme
Large organic molecule
□
Loose bound apoenzyme
□
§
○
•
Enzymes display saturation kinetics
Kcat: measure enzyme turnover
How quick enzyme can convert reactants to products per unit time
○
High Kcat = faster turn over
○
•
Vmax: max observed initial velocity
•
Saturation kinetics: application of increasing substrate concentration
until further increase in substrate has no effect on velocity
•
Line weaver burk: double reciprocal plot
Turn graph into line graph
○
•
michaelis constant (Km): indicates substrate concentration when rxn
rate is 1/2 Vmax
•
Kcat/Km: enzyme efficiency and substrate specificity
Higher value = better
○
•
Analysis of kinetics can distinguish different enzyme mechanism
Random substrate binding: any substrate can bind first
No set order
○
•
Ordered substrate binding: specific order to substrate binding
•
Ping pong mechanism: reactant 1 binds, creates product 1 (may cause
conformational change) then reactant 2 binds and creates product 2
•
Enzyme inhibition
Inhibitors: compound binds to enzyme to prevent enzymatic action
•
Two classes
Reversible (non covalent)
Competitive: bind to active site and block binding of substrate
○
Noncompetitive: substrate and inhibitor bind simultaneously
Common in bi-substrate rxn
§
Km and Vmax = decreased
§
○
Mixed: mix of noncompetitive and competitive
○
•
Irreversible (covalent)
Enzyme will not come off and the effects cant be reversed
○
Inactivate specific target enzyme or class of enzymes
○
Covalent modification use DFP
Bind serine in chymotrypsin
§
○
•
Regulation of enzyme activity
Substrate level of control
Require large change in concentration of substrate
○
Crude means of regulation
○
•
Regulation at commuted steps pathway
Feedback inhibition/activation at control points
○
Mediated allosteric enzymes
○
Maintain homeostasis
○
•
Covalent modifications
Irreversible
○
Reversible
○
Of the Amino acid itself
Note: don’t confuse these with inhibitors
§
○
•
Feedback inhibition
Product E can inhibit enzyme 1 earlier in metabolic pathway
○
•
Reversible covalent modification
Ex. phosphorylation
○
•
Chapter 8: Enzymes
Tuesday, June 5, 2018
7:02 PM
Enzymes and enzyme classes
Enzyme: used by living organisms to accelerate bio rxns
Bio and protein based
○
Lower activation energy of rxn
○
Neither consumed or reduced by rxn
○
•
Classes of enzymes
Oxidoreductases Transferases Hydrolase Lysases
Isomerase
Ligase
Ex. Alcohol
dehydrogenase
Through
reduction/oxidation
Ex. Hexokinase
Take one part of
compound and put it
on the next
Ex. Cleavage of
peptide bond
using H2O
Has to be water
that cleaves the
compound using
hydrolase
Ex. Pyruvate
decarbonylase
Cleave single
compound into
two parts
Ex. Malaeate
Groups are
added.
Compound A is
isomerized to
compound B
Used to
glue to
compounds
together
Enzymes stabilize transition state
Activation energy: energy required by a starting specie to undergo rxn
•
Climb to transition state decreases from non catalyzed to catalyzed
Activation energy decrease from non catalyzed to catalyzed
○
•
Free energy of product and reactant = unchanged
Hess law and state function
○
•
Raising T increase the rate constant by increasing average kinetic energy
of the substrate and increases K
Temp = avg of kinetic energy
○
•
Lock and key model
Lock and key model: substrate fits into enzyme in specific manner
For every lock there is a specific key
○
Specific shape of substrate will fit to enzyme
○
•
Induced fit model: conformational changes in enzyme structure to fit
incoming substrate
Stabilize transition state through conformational change
Help promote rxn
§
○
Favored model
○
•
Enzymatic catalysis proceeds by one of 5 ways
Catalysis: rate enhancement of rxn
For each enzymatic rxn you can use one of the following:
General acid/base catalysis (GABC)
H bond network
□
Series of protonation and deprotonation
□
§
Covalent catalysis
Stabilize a particular transition state
□
§
Electrostatic stabilization
Bring in charged species or high EN specie to stabilize
the transition state
Want to neutralize
®
□
§
Proximity effects
Van der waal
□
§
Preferential stabilization of transition state
Via active site residue
□
Enzyme change shape
□
§
○
•
Protein conformation = role in catalysis
•
Enzymes must stabilize the transition state to achieve rate enhancement
Have different paths that yield the same results
○
•
Environmental effect on enzyme activity
Activity of enzyme depends on the number of local environmental
conditions
•
pH
Important individually
○
Change in pH can change the rate of protonation and deprotonation
○
•
Temperature
•
Ionic strength
Protein solubility
○
•
Substrate/enzyme concentration
•
Inhibitors /activators
•
Each enzyme has optimum set of conditions
Allow peak enzyme activity
○
•
Lysozyme
Cleave polysaccharide
Long chains of NAM and NAG
Cleave between 4th and 5th C on 6C chain
§
○
•
Has cleft
Clamps onto polysaccharide here
○
•
Catalytic mechanism (2 different ones):
Both start and end in the same place
○
Share half chair oxocarbenium ion intermediate
○
•
Philips mechanism
Oxocarbenium ion (+) stabilized by Glu/Asp residue (-)
○
•
Koshland mechanism
Form covalent adduct
No charge stabilization
§
○
•
Lysozyme activity v pH
Lysozyme works well in acidic environments
•
Glu35: must be protonated to act as general acid catalyst in rate
determining step
Want pH under pka of side chain
○
•
Asp52: deprotonated to attack oxonium ion
Has pKa of 3.86
○
pH > 3.6: deprotonate aspartate side chain
○
pH < 3.8: protonate side chain aspartate
○
•
Basically! Glu35 is protonated by Asp52. Asp52 is deprotonated and
makes a good nucleophile which attacks the oxonium ion
•
Protease
Chymotrypsin: serine protease
Enzyme that breaks peptide bonds
○
•
Catalytic triad
Conserved catalytic residue
○
•
Asp (D102) deprotonates His(H57) which then deprotonates Ser (S195).
Ser then nucleophilic attacks the carbonyl carbon on the incoming
polypeptide
•
Mechanism:
Oxyanion hole stabilizes and use residue to stabilize oxygen
○
•
Non protein molecules
Cofactors: non protein compounds
Utilize for enzyme activity
○
Role in enzyme function
○
•
Prosthetic group: non protein group that are tightly bound to enzyme
Not covalent
○
Ex. Heme group
○
Play structural role in enzyme function
○
Stay with enzyme instead of moving between enzyme
○
•
Two categories:
Inorganic (cofactors)
Metals
Common: Fe, Cu, Zn, Mn, Co
□
Rare: Ni, Mo, V, Se
□
Mg: not catalytic
Used for coordination and structure
®
□
§
○
Organic (coenzymes)
Vitamin derived compounds (ex. NADH or FADH)
§
Enzyme associated is transient
§
Cosubstrates when they travel b/w enzymes
§
○
•
Apoenzyme: enzymes w/o cofactor
•
Holoenzyme: with cofactor
•
Overview:
Enzyme
Holoenzyme (complex)
Protein and non protein
§
Apoenzyme: protein part
§
○
Simple: only protein
○
Cofactor
Prosthetic
Small inorganic molecule or atom
□
Tight bound apoenzyme
□
§
Coenzyme
Large organic molecule
□
Loose bound apoenzyme
□
§
○
•
Enzymes display saturation kinetics
Kcat: measure enzyme turnover
How quick enzyme can convert reactants to products per unit time
○
High Kcat = faster turn over
○
•
Vmax: max observed initial velocity
•
Saturation kinetics: application of increasing substrate concentration
until further increase in substrate has no effect on velocity
•
Line weaver burk: double reciprocal plot
Turn graph into line graph
○
•
michaelis constant (Km): indicates substrate concentration when rxn
rate is 1/2 Vmax
•
Kcat/Km: enzyme efficiency and substrate specificity
Higher value = better
○
•
Analysis of kinetics can distinguish different enzyme mechanism
Random substrate binding: any substrate can bind first
No set order
○
•
Ordered substrate binding: specific order to substrate binding
•
Ping pong mechanism: reactant 1 binds, creates product 1 (may cause
conformational change) then reactant 2 binds and creates product 2
•
Enzyme inhibition
Inhibitors: compound binds to enzyme to prevent enzymatic action
•
Two classes
Reversible (non covalent)
Competitive: bind to active site and block binding of substrate
○
Noncompetitive: substrate and inhibitor bind simultaneously
Common in bi-substrate rxn
§
Km and Vmax = decreased
§
○
Mixed: mix of noncompetitive and competitive
○
•
Irreversible (covalent)
Enzyme will not come off and the effects cant be reversed
○
Inactivate specific target enzyme or class of enzymes
○
Covalent modification use DFP
Bind serine in chymotrypsin
§
○
•
Regulation of enzyme activity
Substrate level of control
Require large change in concentration of substrate
○
Crude means of regulation
○
•
Regulation at commuted steps pathway
Feedback inhibition/activation at control points
○
Mediated allosteric enzymes
○
Maintain homeostasis
○
•
Covalent modifications
Irreversible
○
Reversible
○
Of the Amino acid itself
Note: don’t confuse these with inhibitors
§
○
•
Feedback inhibition
Product E can inhibit enzyme 1 earlier in metabolic pathway
○
•
Reversible covalent modification
Ex. phosphorylation
○
•
Chapter 8: Enzymes
Tuesday, June 5, 2018
7:02 PM
Enzymes and enzyme classes
Enzyme: used by living organisms to accelerate bio rxns
Bio and protein based
○
Lower activation energy of rxn
○
Neither consumed or reduced by rxn
○
•
Classes of enzymes
Oxidoreductases Transferases Hydrolase Lysases Isomerase Ligase
Ex. Alcohol
dehydrogenase
Through
reduction/oxidation
Ex. Hexokinase
Take one part of
compound and put it
on the next
Ex. Cleavage of
peptide bond
using H2O
Has to be water
that cleaves the
compound using
hydrolase
Ex. Pyruvate
decarbonylase
Cleave single
compound into
two parts
Ex. Malaeate
Groups are
added.
Compound A is
isomerized to
compound B
Used to
glue to
compounds
together
Enzymes stabilize transition state
Activation energy: energy required by a starting specie to undergo rxn
•
Climb to transition state decreases from non catalyzed to catalyzed
Activation energy decrease from non catalyzed to catalyzed
○
•
Free energy of product and reactant = unchanged
Hess law and state function
○
•
Raising T increase the rate constant by increasing average kinetic energy
of the substrate and increases K
Temp = avg of kinetic energy
○
•
Lock and key model
Lock and key model: substrate fits into enzyme in specific manner
For every lock there is a specific key
○
Specific shape of substrate will fit to enzyme
○
•
Induced fit model: conformational changes in enzyme structure to fit
incoming substrate
Stabilize transition state through conformational change
Help promote rxn
§
○
Favored model
○
•
Enzymatic catalysis proceeds by one of 5 ways
Catalysis: rate enhancement of rxn
For each enzymatic rxn you can use one of the following:
General acid/base catalysis (GABC)
H bond network
□
Series of protonation and deprotonation
□
§
Covalent catalysis
Stabilize a particular transition state
□
§
Electrostatic stabilization
Bring in charged species or high EN specie to stabilize
the transition state
Want to neutralize
®
□
§
Proximity effects
Van der waal
□
§
Preferential stabilization of transition state
Via active site residue
□
Enzyme change shape
□
§
○
•
Protein conformation = role in catalysis
•
Enzymes must stabilize the transition state to achieve rate enhancement
Have different paths that yield the same results
○
•
Environmental effect on enzyme activity
Activity of enzyme depends on the number of local environmental
conditions
•
pH
Important individually
○
Change in pH can change the rate of protonation and deprotonation
○
•
Temperature
•
Ionic strength
Protein solubility
○
•
Substrate/enzyme concentration
•
Inhibitors /activators
•
Each enzyme has optimum set of conditions
Allow peak enzyme activity
○
•
Lysozyme
Cleave polysaccharide
Long chains of NAM and NAG
Cleave between 4th and 5th C on 6C chain
§
○
•
Has cleft
Clamps onto polysaccharide here
○
•
Catalytic mechanism (2 different ones):
Both start and end in the same place
○
Share half chair oxocarbenium ion intermediate
○
•
Philips mechanism
Oxocarbenium ion (+) stabilized by Glu/Asp residue (-)
○
•
Koshland mechanism
Form covalent adduct
No charge stabilization
§
○
•
Lysozyme activity v pH
Lysozyme works well in acidic environments
•
Glu35: must be protonated to act as general acid catalyst in rate
determining step
Want pH under pka of side chain
○
•
Asp52: deprotonated to attack oxonium ion
Has pKa of 3.86
○
pH > 3.6: deprotonate aspartate side chain
○
pH < 3.8: protonate side chain aspartate
○
•
Basically! Glu35 is protonated by Asp52. Asp52 is deprotonated and
makes a good nucleophile which attacks the oxonium ion
•
Protease
Chymotrypsin: serine protease
Enzyme that breaks peptide bonds
○
•
Catalytic triad
Conserved catalytic residue
○
•
Asp (D102) deprotonates His(H57) which then deprotonates Ser (S195).
Ser then nucleophilic attacks the carbonyl carbon on the incoming
polypeptide
•
Mechanism:
Oxyanion hole stabilizes and use residue to stabilize oxygen
○
•
Non protein molecules
Cofactors: non protein compounds
Utilize for enzyme activity
○
Role in enzyme function
○
•
Prosthetic group: non protein group that are tightly bound to enzyme
Not covalent
○
Ex. Heme group
○
Play structural role in enzyme function
○
Stay with enzyme instead of moving between enzyme
○
•
Two categories:
Inorganic (cofactors)
Metals
Common: Fe, Cu, Zn, Mn, Co
□
Rare: Ni, Mo, V, Se
□
Mg: not catalytic
Used for coordination and structure
®
□
§
○
Organic (coenzymes)
Vitamin derived compounds (ex. NADH or FADH)
§
Enzyme associated is transient
§
Cosubstrates when they travel b/w enzymes
§
○
•
Apoenzyme: enzymes w/o cofactor
•
Holoenzyme: with cofactor
•
Overview:
Enzyme
Holoenzyme (complex)
Protein and non protein
§
Apoenzyme: protein part
§
○
Simple: only protein
○
Cofactor
Prosthetic
Small inorganic molecule or atom
□
Tight bound apoenzyme
□
§
Coenzyme
Large organic molecule
□
Loose bound apoenzyme
□
§
○
•
Enzymes display saturation kinetics
Kcat: measure enzyme turnover
How quick enzyme can convert reactants to products per unit time
○
High Kcat = faster turn over
○
•
Vmax: max observed initial velocity
•
Saturation kinetics: application of increasing substrate concentration
until further increase in substrate has no effect on velocity
•
Line weaver burk: double reciprocal plot
Turn graph into line graph
○
•
michaelis constant (Km): indicates substrate concentration when rxn
rate is 1/2 Vmax
•
Kcat/Km: enzyme efficiency and substrate specificity
Higher value = better
○
•
Analysis of kinetics can distinguish different enzyme mechanism
Random substrate binding: any substrate can bind first
No set order
○
•
Ordered substrate binding: specific order to substrate binding
•
Ping pong mechanism: reactant 1 binds, creates product 1 (may cause
conformational change) then reactant 2 binds and creates product 2
•
Enzyme inhibition
Inhibitors: compound binds to enzyme to prevent enzymatic action
•
Two classes
Reversible (non covalent)
Competitive: bind to active site and block binding of substrate
○
Noncompetitive: substrate and inhibitor bind simultaneously
Common in bi-substrate rxn
§
Km and Vmax = decreased
§
○
Mixed: mix of noncompetitive and competitive
○
•
Irreversible (covalent)
Enzyme will not come off and the effects cant be reversed
○
Inactivate specific target enzyme or class of enzymes
○
Covalent modification use DFP
Bind serine in chymotrypsin
§
○
•
Regulation of enzyme activity
Substrate level of control
Require large change in concentration of substrate
○
Crude means of regulation
○
•
Regulation at commuted steps pathway
Feedback inhibition/activation at control points
○
Mediated allosteric enzymes
○
Maintain homeostasis
○
•
Covalent modifications
Irreversible
○
Reversible
○
Of the Amino acid itself
Note: don’t confuse these with inhibitors
§
○
•
Feedback inhibition
Product E can inhibit enzyme 1 earlier in metabolic pathway
○
•
Reversible covalent modification
Ex. phosphorylation
○
•
Chapter 8: Enzymes
Tuesday, June 5, 2018 7:02 PM
Document Summary
Enzyme: used by living organisms to accelerate bio rxns. Take one part of compound and put it on the next. Has to be water that cleaves the compound using hydrolase. Activation energy: energy required by a starting specie to undergo rxn. Climb to transition state decreases from non catalyzed to catalyzed. Activation energy decrease from non catalyzed to catalyzed. Free energy of product and reactant = unchanged. Raising t increase the rate constant by increasing average kinetic energy of the substrate and increases k. Lock and key model: substrate fits into enzyme in specific manner. For every lock there is a specific key. Specific shape of substrate will fit to enzyme. Induced fit model: conformational changes in enzyme structure to fit. Used to glue to compounds together: malaeate roups are dded. Induced fit model: conformational changes in enzyme structure to fit incoming substrate. Enzymatic catalysis proceeds by one of 5 ways.
