72
142
An Introduction to Enzyme and Coenzyme Chemistry, 2nd Ed.T. D. H. Bugg, Blackwell Science, Oxford, 2004
Proteases!!!Phosphatases
Thiamin!!Pyridoxal phosphate!!Nicotinamide!!!Flavin coenzyme!!Heme!!!!Vitamin B12
Hydrolytic Enzymes:
Cofactors (Coenzymes):
Chapter 5, pp 81-98Chapter 5, pp 102-109
Chapter 7, pp 176-179Chapter 9, pp 210-221Chapter 6, pp 121-129Chapter 6, pp 129-142Chapter 6, pp 143-147Chapter 11, pp 240-244
2 e-
1 e-
1 e- or 2 e-redox
mutase
143
Enzymes: all enzymes are proteinscatalysts speed up reactions by lowering the activation energy
(ΔG‡), NOT by changing the thermodynamics of the reaction (ΔG°)
Stabilization of the transition stateBringing reactants together in the proper orientation for the reaction
to occur
73
144
H3C O
OBr
CH3CO2-+
H3C -O
O Br
O CH3
O
+Krel = 1 M-1 s-1
O
OBr
CO2-
O
O
O- ArO-Krel = 220 s-1 effect molarity 220 M
O
OBr
-O2C
O
O
O
Krel = 5 x 104 s-1 effect molarity 5 x 104 M- ArO-
O
OBr
O
O
O
- ArO-
O
OBr
-O2C
CO2-
Krel = 2 x 106 s-1 effect molarity 2 x 106 M
O
CO2-
O
O
Br
- ArO-O O
O
O
Krel = 1 x 107 s-1 effect molarity 1 x 107 M
145
O
OBr
-O2C
O
OBr
-O2C
O
OBr
CO2-
reactive conformation for cyclization reaction
O
OBr
CO2-
held (pre-organized) inthe reactive conformation
Krel = 5 x 104 s
-1
Krel = 2 x 106 s
-1
74
146
Mechanism:
Mechanisms can not be proven absolutely, they can onlybe disproved (inconsistent with the available data)
Accepted mechanism is based on the preponderance ofthe available evidence
• consistent with the products- labeling studies• intuition: consistent with well-known chemistry and
widely accepted mechanistic tenets • consistent with modest changes in the substrate• kinetic rate expression• model reactions- easier to study
147
Proteases: catalyzes the hydrolysis of peptide bondsBugg, Chapter 5, pp. 81-98
NH
HN
NH
O
RO
HN
N
O R
RO
R
H
O protease
H2OH3N
CO2 NH
HN
H3N
O
RO
HN
N
O R
RO
R
H
O
H3NCO2O +
1. Serine protease Bugg, p. 842. Cysteine protease p. 893. Aspartyl protease p. 954. Zinc (metallo) protease p. 92
•chymotrypsin: cleaves at the C-terminal sideof aromatic residues Phe, Tyr, Trp
• trypsin: cleaves at the C-terminal side of basic residues Arg, Lysbut not His
75
148
Serine Protease: Chymotrypsin
Bugg, p. 96
OSer195
H
N
NHis57
R1 NH
O
R2
NH HN
oxy-anionhole
H
Asp102 CO2-
OSer195
H
N
NHis57
H
Asp102 CO2-
H2OR1CO2H
R2-NH2
+
149
Ser-195
His-57
Asp-102
Catalytic triad of α-chymotrypsin
pdb code: 5CHA
76
150
Active site of bovine trypsin with a bound inhibitor
Bugg, p. 85Katz, B. A.; Finer-Moore, J.; Mortezaei, R.; Rich, D. H.; Stroud, R. M.
Biochemistry 1995, 34, 8264-8280.
Ser-195
His-57
Asp-102
Asp-189
HN
NH
HN B
O
tBuO
O
O
NH3
OEt
OEt HN
NH
HN B
O
tBuO
O
O
NH3
OEtEtO
O Ser-175
pdb code: 1BTX
151
Ser-195
His-57Asp-102
His-57
Asp-102
Gly-196
Gly-196 Ser-195Gly-193
Gln-192
Gln-192
Gly-193Asp-194
Asp-194
Oxy-anion hole of trypsin
HN
NH
HN B
O
tBuO
O
O
NH3
OEtEtO
O Ser-175
pdb code: 1BTX
77
152
Cysteine Protease: Papain (212 amino acids)active-site cysteine and histidine, overall mechanism is
similar to serine proteasesusually not a digestive enzyme (intracellular)
Bugg, p. 91
SCys25
H
N
NHis159
H
R1CO2H
+
R2-NH2
+SCys25
H
N
NHis159
R1 NH
O
R2
NH HN
oxy-anionhole
H
H2O
153
O NH
HN
NH
O
O
O
H
O
O NH
HN
NH
O
O
OH
O
S Cys25
NH
O
Asn19
N
H
H
papain
oxyanionhole
His157
Cys25
Asn19
pdb code: 1BP4
oxyanionhole
Structure of papain with a bound inhibitor
78
154
Aspartyl Protease Mechanism: Renin, HIV proteaseBell shaped pH vs. rate profile: max rate at pH ~ 2.4 indicative of general acid-general base catalysis.
RN
H
O
R'
Asp
OO
H O
O
Asp
O H
H
_
Asp
OHO
RN
H
H HO R'
O
O
O
Asp
_
155
HIV Protease: an Aspartyl Protease
with a bound inhibitor
N
O
CH3
N
O
H OPh
OH
N
H
O
N
H
N
O
CH3
inhibitor
Ph
Catalytic Asp-25
pdb code: 1HVJ
79
156
N
O
CH3
N
O
H OPh
OH
N
H
O
N
H
N
O
CH3
inhibitor
Ph
Interaction of the catalytic Asp-25 with the bound inhibitor
pdb code: 1HVJ
157
General base mechanism
Nucleophilic mechanism (acyl enzyme complex)
Metallo-protease (zinc): carboxypeptidase:important catalytic groups: Glu-270, Tyr-248Zn ion coordination: Glu-72, His-196, His-69
Bugg, p. 94
80
158
Active site of carboxypeptidase
pdb code: 2CTC
Glu-270
His-69Tyr-248
His-196
Glu-72
Bound ligand
ZnH2O
Arg-145
159
Phosphate ester hydrolysis
1. Phosphatases: over transfer (hydrolysis) of a phosphate monoester to water
2. Phosphodiesterases: hydrolysis of a phosphodiester toa phosphate monoester and an alcohol
3. Kinases: transfer of the γ-phosphate group of ATP to an acceptor group
Glucose 6-phosphatase
HO
O3PO
HOHO
OH
-2
glucose-6-phosphate
HO
HO
HOHO
OH
+ H2O
- H2O+ PO4 -3
(Pi)
CH2OPO3
OH
HO
HOO
O3PO-2
-2
fructose-1,6-bisphosphate
CH2OH
OH
HO
HOO
O3PO-2
+ PO4 -3
(Pi)
+ H2O
- H2O
Fructose 1,6-bisphosphatase: M+2 dependent
Alkaline phosphatase: M+2 dependentBugg, pp. 102-109
81
160
Phosphatases: General acid-base mechanism
161
Phosphatases: Covalent mechanism
SN2 mechanism
82
162
Glucose 6-phosphatase: covalent catalysis
Fructose 1,6-bisphosphatase: General acid-base catalysis
163
Associative vs dissociative mechanism: Chiral phosphateUse three isotopes of oxygen, 16O, 17O, 18O
RO P
S
OO
__
+ H2O
ROH O P
S
OO
_
__
+
R1O P
O
OO
__
+R2OH
R1OH R2O P
O
OO
_
__
+OR2P
O
OO _
_
OP
S
OO
_
__
-or-
-or-
Does the observation of stereochemically defined products (retention or inversion of stereochemistry) rule out thedissociative mechanism involving metaphosphate ion?
83
164
Tyrosine phosphataseconserved aspartate, cysteine, and arginine
Tyr OTyr OH + PO4 -3
(Pi)
+ H2OP
O
OO
__
N
HN
N
H
HH
H Arg
+
Asp
OO
H
S Csy_
165
Phosphodiesterase: Ribonuclease (RNase): catalyzes the hydrolysis of the
phosphodiester bond of RNA. Catalytic groups are His-12 and His-119
Bell shaped pH vs. rate profile: max rate at pH ~ 6.8 indicative of general acid-general base catalysis. Mechanism requires both an imidazole and imdiazolium for catalysis
pKa of hisidine is ~ 6.1
Bugg, pp. 104-109
B
O
OHO
O
PO
-O
B
O
OO
O
H
NH
NHis
H
N NH
His
RNase
B
O
OHO
HO
PO
-O
B
O
OHO
O
O-
NH
NHis
H
N NH
His
84
166
Some Cofactors
OP
OHO
HO
N
O
OH
H
N N
SO
P
HO O
O POH
OH
O
NH2
N S
HN
NH
O
OH
O+
Thiamin Diphosphate Pyridoxal PhophatesBiotin
N
OH
OHN
OH
NO
O
HN
O
PO
O
O-
PO
O
O-
O
HO
N
OH
N
N
NH2N
Na+Na+
N
NN
N
O
HOO
OH
Fe
FAD
Heme
HO
ON
OH
P
O
OH
O P
O
OH
OHO
OO
N
NN
N
NH2
O
R
N
OH
OHN
OH
NO
O
HN
O
PO-
O
O-
FMN
R= H NADHR= -PO3H2 NADPH
O
NH2
N
N
N
NH2
N
O
OP
OH
O
O
OPO
OH
OH
O
HN
HN
O
SH
OHO
OH
PHO
O
Coenzyme A (CoA)
NN
N N
H2N
O NH2
O
NH2
NH2O
O
O
Co
O
H
H2N
O
NH
O
C
PO
O
-OO
N
N
HO H
H
NH2
H
H
OH
N
Vitamin B12
O
NH
N
N
N
NH2
NH
NH
O
O
HO
O
OH
SS
H
OH
O
Folic Acid
Lipoic Acid
HN
O
HS
O
NH
NH2
HO
O OH
O
Glutathione
167
Thiamin Dependent Reactions (Vitamin B1)(Bugg, Chapt. 7, pp. 176-9)
Acetolactate synthase
CH3C
O
CO2H2
thiamin
CH3C
O
C
OH
CH3
CO2H+ CO2
Pyruvate dehydrogenase: pyruvate decarboxylase, dihydrolipoyl transacetylase, dihydrolipoyl dehydrogenase,
CH3C
O
CO2H
pyruvate
+ thiamin + lipoic acid + CoA-SH Acetyl-CoA + CO2(CH3COS-CoA)
N N
SO
P
-O O
O PO-
O-
O
NH2
N
+
Thiamin Diphosphate (pyrophosphate)
SH
NH
OSH3C
O
Lys
S
NH
OLys
S
CO2-
O
- CO2
thiamin, CoA-SH
S-CoA
O
Pyruvate Decarboxylase
CH3C
O
CO2H
thiamin
CH3C
O
H + CO2
85
168
Mechanistic insight into thiamin dependent reactions:Breslow, R. J. Am. Chem. Soc. 1958, 80, 3719.
Benzoin condensation
pKa measured tobe between 12-18
N N
SO
P-O O
O PO-
O-
O
NH2
N
+
H D2ON N
SO
P-O O
O PO-
O-
O
NH2
N
+
D
t1/2 ~ 20 min
2 PhCHO + - CN + - CN
pKa ~ 9.3Ph
OH
Ph
O
169
Mechanism of pyruvate decarboxylase:
Mechanism of acetolactate synthase:
N
SPPO H
R
CH3C
O
CO2H+
86
170
Pyruvate Dehydrogenase: dihydrolipoyl transacetylase
171
Transketolase: carbohydrate biosynthesisH2C
O
H OH
H2C
H OH
OPO3
H
CHO
OH
H OH
H2C
H OH
OPO3
D-ribose-5-P (C5 aldose)
OH
+
D-ribulose-5-P (C5 ketose)
H2C
O
HO H
H OH
H OH
OH
OHH
H2C OPO3
sedohepulose-7-P (C7 ketose)
H
CHO
OH
H2C OPO3
glyceraldehyde-3-P (C3 aldose)
+
thiamin-PP
87
172
Thiamin acidity and anion stability is special to the N-alkyl thiazole ring system: anion is stabilized by three major
resonance strictures: two are ylides, one is a carbene
Nature may have tried the imidazole and oxazole ring systems, but these would not have performed the necessary chemistry.
SNRSNR
ylide carbene
SNR
central carbonhas 6 electrons
ylide
NNR
H
Not acidic
ONR
H
more acidic than thiamin, but the anion is hydrolytically labile
173
Pyridoxal Phosphate (PLP) dependent enzymes (Vitamin B6) (Bugg, Chapt. 9, pp 210-221)
Involved in amino acid biosynthesis, metabolism and catabolism
N
2-O3PO
OH
O H
N
2-O3PO
OH
NH2
pyridoxal
N
2-O3PO
OH
OH
pyridoxamine pyridoxine
R
NH3
CO2-
H
L-amino acid
R
NH3
HCO2
-
D-amino acid
racemase,epimerase
R
NH3
HH
decarboxylase
R
O
CO2-
transaminase
R
NH2
CO2-
H2O
R'
NH3
CO2-
H
reaction at the! or " carbon
#
!
88
174
Pyridoxal is often covalently bound to the resting enzyme as a Schiff base to the sidechain of an active site lysine residue.
NH
PO
N
O
Lys
H
175
Decarboxylase:HO
HO
CO2
NH3
HO
HO
NH3
L-DOPAdecarboxylase
NH
HO NH3
CO2
NH
HO NH3
Serotonin
Dopamine
89
176
Transaminase: amino acid biosynthesis
177
Stereochemistry of the transamination reaction:proton transfer is mediated by an active site lysine
NH
PO
NH2
OHR CO2
-
O
+
H
H
NH
PO
N
O
H
R CO2-
H
H
NH
PO
N
O
CO2-
H
R
H
H
NH
N
H
OH-O2C
R
OP
Enzyme
H2N
H
NH
N
H
OH-O2C
R
OP
Enzyme
H2N
H
NH
N
H
H
OH-O2C
R
OP
Enzyme
H2N
90
178
Stereochemistry of transamination (aspartate aminotransferase)
NH
N
H
OH-O2C
OPO3-
Enzyme
H2N
HNH
N
H
OH-O2C
OPO3-
Enzyme
H2N
H
NH
N
H
H
OH-O2C
OPO3-
Enzyme
H2N
-O2C -O2C -O2C
NH2H2N
HN Arg
NH2H2N
HN Arg
NH2H2N
HN Arg
H2NNH2
HN
Arg
NH2H2N N
H
Arg
H2NNH2
HN
Arg
NH2H2N N
H
Arg
NH2H2N N
H
Arg
H2NNH2
HN
Arg
Arg-386
Lys-258
Arg-292
Arg-266
Pdb code: 1AJS
179
Reactions at the β- and γ-carbons of amino acids
H3C
NH3
CO2-
H
!
"OH
Threonine
threoninedehydratase
H3C
O
CO2-
+ NH4
HO
NH3
CO2-
H
NH3
CO2-
serine hydroxymethyltransferase
+ O
H
H" "
formaldehydeequivalent
HO
NH3
CO2-
H
NH
+
NH
NH3
CO2-
Htryptophansynthase
NH3
CO2-
HH3CS
methioninelyase,H2O
H3C
O
CO2-
+ NH4 + CH3SH
+ H2O
!
"
#
91
180
Threonine Dehydratase
181
Tryptophan Synthase:reactivity of indole toward electrophiles:
Similar to the mechanism of threonine dehyratase an electophilicPAL-amino acid complex is generated
NH
PO
N
O
CO2-
H
H
Enzyme
:B
OH
Enzyme
B H
92
182
Tryptophan Synthase (con’t):
183
Serine hydroxymethyl transferase:one-carbon (methyl) donors in biology
HO
NH3
C CO2-
HHH
PAL, Folic acid
2 NADPH
HN
N
N
N
H2C N
H2N
O
HN
O
CO2-
CO2-
methylene tetrahydrofolate
2 NADPHHN
N
N
N
HN
H2N
O
HN
O
CO2-
CO2-
Folic acid
HN
N
NH
HN
HN
H2N
O
HN
O
CO2-
CO2-
NH3
CO2-
HH3CS
O N
N
N
N
NH2
HO OH
OPO
O-
O
P
O
O
O-
P-O
O-
O
ATP
+
O N
N
N
N
NH2
HO OH
S
H3C
H3N
CO2-
H
S-adenosyl methionine (SAM)
- PO4-3, - P2O7
-4
Bugg, Ch. 5, pp. 220-1
93
184
Methyl groups from:
SAM
OH3C
H3COH
H3C
OH
O
CH3
H3C
O
O
CH3
O
CH3
OH
O
O
HO N
CH3
H3C
CH3
OH
OCH3
H3C
CH3
CH3O
O
O
OH
OH
O
O
OH
OH3C
OHNH2
Erythromycin Daunomycin
N N
CH3OO
NHH3C
CH3 CH3
Physostigmine
Methylene tetrahydrofolate
O
HO
2-O3PO N
NH
O
O
H3C
185
Mechanism of serine hydroxymethyl transferase:
Note: this mechanism is not the same as on page 221 of Bugg, which is probably incorrect
94
186
Mechanism of methionine γ-lyase
NH
PO
N
O
CO2-
H
H
Enzyme
:BH3CS
