Review
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Enzyme “constants”
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Reversible inhibition
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Km
Vmax
kcat
kcat/Km
Ki
E+S+I
ES + I
EI
ESI
Impact on Km and Vmax for each
Irreversible inhibition
–
I combines/binds to E to form a very
stable complex
E+P
Question….
• Methanol (wood alcohol) is highly toxic because
it is converted to formaldehyde in a reaction
catalyzed by the enzyme alcohol
dehydrogenase:
• NAD+ + methanol  NADH + H+ + formaldehyde
• Based on enzyme inhibition, what’s a possible
treatment for methanol poisoning?
Irreversible inhibition
• Suicide/mechanism based inhibitor
– A few chemical steps are carried out
– Compound converted to reactive intermediate that
irreversibly reacts with enzyme
– Used in drug design
• Potential for high potency
• Typically very specific for the enzyme: few side effects
Chymotrypsin: Specific enzyme mechanism
• Protein structure
determines function
Chymotrypsin
• Protease specific for bonds adjacent to aromatic
AA
• Hydrolysis reaction
– But: enzyme doesn’t catalyze direct attack by water
• Stabilization of E-TS
• General acid/base and covalent catalysis
• Two phases to the reaction:
1. Acylation
Cleavage of peptide bond and formation of ester
with enzyme
2. Deacylation
Hydrolysis of ester and enzyme regenerated
Kinetics → Mechanism
Fast phase (burst phase/pre-steady state)
Kinetics → Mechanism
Histidine must
be deprotonated
for rxn to occur
Ile (N-term) must be
protonated for substrate
to bind
Chymotrypsin: “Catalytic triad”
protease
• Catalytic triad
components
– Nucleophile-Ser195
– His57 (general base)
– Asp102 (stabilizes +
charge on His)
• Oxyanion hole
– Stabilizes O- in
tetrahedral
intermediate
Chymotrypsin
Chymotrypsin mechanism
Chymotrypsin mechanism
Chymotrypsin
• Formation of acyl-enzyme intermediate
– Covalent bond between enzyme and
substrate/transition state
• Actual breaking of the peptide bond
• Deacylation
– Activation of water to break the enzymesubstrate bond
• Release of the rest of the substrate protein
Enzymes and regulation
•
Activity can modulated by several factors
–
Maximize biological efficiency: stop or speed-up a
pathway under appropriate conditions
1. Allostery
2. Reversible covalent modification
–
Addition of sugars, phosphates, adenine, acetate, etc.
3. Reversible binding of other, regulatory, proteins
4. Proteolytic cleavage
Allostery
• Modulation of equilibrium between
more/less active forms
Allostery
• Aspartate transcarbamoylase
– Pyrimidine synthesis
• Binding of modulator to regulatory
subunit inhibits activity
• CTP negative modulator
• Feedback (product inhibition)
Allostery: a case where M-M doesn’t
quite work
Sigmoidal V vs S curves
Change in Vmax, not K0.5
Change in K0.5, not Vmax
Covalent modification
• Phosphorylation, adenylation, uridylation,
methylation…..
– Change electrostatic interactions/repulsion
• Phosphorylation of serines adds a negative charge
• Acetylation of lysines removes a positive charge
– Conformational change → turns ‘on’ or ‘off’
Reversible phosphorylation
• Phosphate added by kinases
• Phosphate removed by phosphatases
• Typically serine/threonine or tyrosine,
sometimes histidine
Phosphorylation of glycogen
phosphorylase
• Glycogen
phosphorylase ‘a’
and ‘b’
– Converts glycogen
to glucose 1phosphate for
energy
Proteolytic cleavage
• Synthesis as ‘zymogen’
(inactive enzyme precursor)
– Chymotrypsinogeninactive →
chymotrypsinactive
– Cleavage → conformational
change that exposes active site
– Mechanism used for other
proteins as well
• Procollagen collagen
• Fibrinogen  fibrin
• Proinsulin insulin