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Enzymes
Most biological catalysts are ____________
(some REALLY COOL ONES are folded RNAs!!!)
Catalysts Catalyst does not alter equilibrium
Enzyme
Nonenzymatic
reaction rate (s-1)
Enzymatic
reaction rate (s-1)
Rate enhancement
Carbonic
anhydrase
1.3 x 10-1
1 x 106
7.7 x 106
Triose phosphate
isomerase
4.3 x 10-6
4300
1 x 109
Staphlococcal
nuclease
1.7 x 10-13
95
5.6 x 1014
Enzymes
E + S
ES
E + P
Highly specific
Reaction occurs in ___________ of enzyme
Substance acted upon = __________
Resulting species = _____________
Enzyme acts on forward and reverse reactions
Activity depends on protein’s native structure
Regulated - by concentrations of substrate and substances other
than substrate
Enzymes
Cofactors
Functional groups of protein enzymes are involved in acid-base reactions,
covalent bond formation, charge-charge interactions
BUT they are less suitable for oxid-reduc and group-transfer reactions
SO they use __________________ (inorganic ions)
COFACTORS
may be metal ions (Cu2+, Fe3+, Zn2+)
trace amounts of metal needed in our diets
Enzymes
Cofactors
COFACTORS can be organic or metalloorganic molecules -->
COENZYMES
Examples:
NAD+
Heme
Holoenzyme =
Apoenzyme (inactive) + cofactor/coenzyme/metal ions
Enzymes
Coenzymes
Coenzymes must be regenerated
Many vitamins are coenzyme precursors
Vitamins must be present in our diets because we cannot synthesize
certain parts of coenzymes
Coenzyme
Reaction mediated
Vitamin source
Human
Disease
Cobalamin
coenzymes
Alkylation
Cobalamin (B12)
Pernicious anemia
Flavin
coenzymes
Oxidation-reduction
Riboflavin (B2)
rare
Nicotinamide
coenzymes
Oxidation-reduction
Nicotinamide (niacin)
Pellagra
Pyridoxal
phosphate
Amino group transfer
Pyridoxine (B6)
rare
Folic acid
Megaloblastic anemia
Thiamine (B1)
Beriberi
Tetrahydrofolate One-carbon group transfers
Thiamine
pyrophosphate
Aldehyde transfer
Enzymes
Substrate specificity
Types of complementarity between enzyme and substrate:
Substrate binding sites undergo conformational change when substrate binds
Enzymes
Enzyme undergoes conformational change when substrate binds induced fit
Substrate
a
c
b
a
+
b
ES complex
a
b
Enzyme
c
c
Enzyme-substrate complementarity
Dihydrofolate reductase-NADP+(red)-tetrahydrofolate (yellow)
Enzymes
Stereospecific
Why?
Inherently chiral (proteins only consist of L-amino acids) so form asymmetric
active sites
Example: Protein enzyme Yeast Alcohol dehydrogenase (YADH)
YADH
CH3CH2OH + NAD+
Ethanol
O
CH3CH + NADH + H+
Acetaldehyde
Enzymes
Stereospecific
Yeast Alcohol dehydrogenase (YADH) is stereospecific
1. If YADH reaction uses deuterated ethanol, NAD+ is deuterated to form
NADD
O
D H
NADD
O
+
NAD
C
H
YADH
NH2
C
NH2
N
+
N
O
R
R
+ CH3CD + H+
+ CH3CD2OH (ethanol)
(acetaldehyde)
2. Isolate NADD and use in reverse reaction to reduce normal acetaldehyde,
deuterium transferred from NADD to acetaldehyde to form ethanol
D
H
O
C
OH
YADH
Hpro-S
NH2
C
Dpro-R
N
R
CH3
O
+NAD+
+ CH3CH + H+
3. Enantiomer of ethanol - none of deuterium is transferred from this isomer
of ethanol to NAD+ in the reverse reaction
OH
Dpro-S
C
CH3
Hpro-R
Enzyme activity
Dependent on:
[metal ion], pH, temperature, [enzyme], [substrate]
Enzymes
E + S
ES
E + P
G’˚ < 0; favorable
Enzymes
Enzymes affect reaction rates, not ____________
Catalysts enhance reaction rates by lowering __________________
Rate is set by activation energy G‡
Higher activation energy --> _____________
Overall rate of reaction is determined by step with highest
activation energy --> rate-limiting step
Enzymes
General acid-base catalysis
General acid catalysis - partial proton transfer from an acid lowers free energy of
reaction’s transition state
Keto
Transition state
R
Enol
R
C
O
H
CH2
A
R
+
H
O
C
A
C
CH2-
H
H
H
O
H
CH2
A-
+
General base catalysis - partial proton abstraction by a base lowers free energy of
reaction’s transition state
Keto
Transition state
R
R
C
C
O
O
CH2
CH2-
H
H
B
B
Enol
+
+
R
C
H+
O
H
CH2
H
B
Enzymes
General acid-base catalysis
Enzymes
General acid-base catalysis
Example: Ribonuclease A (RNase A)
digestive enzyme secreted by pancreas into small intestine
hydrolyzes RNA
rate depends on pH, suggesting involvement of ionizable residues
His12 and His119
Enzymes
Covalent Catalysis
Transient covalent bond formed between E and S
Accelerates reaction rate through transient formation of a catalyst-substrate
covalent bond
Usually covalent bond is formed by the reaction of a nucleophilic group on the
catalyst with an electrophilic group on the substrate --> nucleophilic catalysis
SA-SB + N:
SA -N + SB
SA + N: + SB
H2O
Example: Decarboxylation of acetoacetate (catalyst contains primary amine)
O
CH3
C
acetoacetate
CH2
acetone
O
O
C
CH3
O-
C
RNH2
+ RNH2
+ OH-
OHR
CH3
+
N
C
CO2
H
O
CH2
SCHIFF BASE
(IMINE)
CH3
C
O-
R
..
N
CH3
C
H
+ H+
CH2
R
+
N
CH3
C
H
CH3
Enzymes
Covalent Catalysis
Some amino acids with nucleophilic groups
ROH
RSH
RNH3+R
HN
+
NH
Serine
Cysteine
Lysine
Histidine
Enzymes
Metal Ion Catalysis
One-third of all known enzymes require metal ions --> metalloenzymes
Fe2+, Fe3+, Cu2+, Zn2+, Mn2+, Co2+ (sometimes Na+, K+, Mg2+, Ca2+)
Metal bound to enzyme (or substrate)
What can it do?
help orient substrate (or enzyme) for reaction
stabilize charged reaction transition state
mediate oxidation-reduction reactions (change metal’s oxidation state)
Voet, p. 295 11-11, scheme
Enzymes:
Chymotrypsin
Serine protease, very reactive serine residue in enzyme
Digestive enzyme synthesized by pancreas
Catalyzes cleavage of peptide bonds adjacent to aromatic amino acids
Transition state stabilization
General acid-base catalysis and covalent catalysis
Catalytic triad = Ser195, Asp102, His57
Enzymes:
Chymotrypsin
general base
general acid
general acid
Covalent intermediate
general base
Enzymes:
Chymotrypsin
Enzymes:
Chymotrypsin
Enzymes:
Chymotrypsin
Enzymes:
Chymotrypsin
Enzymes:
Chymotrypsin and other serine proteases
Enzymes:
Enolase
catalyzes reaction step of glycolysis
reversible dehydration of 2-phosphoglycerate to phosphoenolpyruvate
Metal ion catalysis, general acid-base, transition state stabilization
Lys345 = general base, abstracts proton from C-2 of 2-phosphoglycerate
Glu211 = general acid, donates proton to -OH leaving group
Enzymes:
Enolase
Metal ion catalysis
2 Mg2+ ions interact with 2-phosphoglycerate making the C-2 proton
more acidic (lower pKa) and easier to abstract