Lecture 9 - Co-factors Part II
PLP
- Alanine Racemase
H Me
OH
H 2N
Designing a mechanism based PLP inhibitor?
PLP.E
OH
H 2N
OH
H 2N
Cl
H
O
Cl
O
BH +
O
-How to inhibit the conversion of L-Ala to D-Ala?
O
O
R H
R
O
O
NH
NH
N
H
H
O
O
D-Ala
O
L-Ala
Cl
Use:
H Me
NH
-Product
-Reactant State
O
NH
N
H
E
N
H
E
NH O
O
NH 2
OH
O
BH +
NH
NH
N
H
N
H
N
H
Another covalent inhibitor:
E
NH 2
O
O
O
O
NH
NH
N
H
N
H
or alternatively:
O
O
H
E
NH 2
NH
inactive covalent adduct
N
H
O
O 2C
NH
O 2C
O
inactive covalent adduct
kinact ~ Partitioning
~
k cat
N
H
Thiamine pyrophosphate (Vitamin B1 )
Like PLP, the electron-deficient heterocyclic ring of thiamine
Reactions involving thiamine pyrophosphate:
1. Decarboxylation of alpha-ketoacids: pyruvate decarboxylase
pyrophosphate (TPP) stabilizes the formation of carbanionic
species.
O
O
H 2N
S
N
O
O
O
P
O
P
O O
O
O
N
N
D 2O
H
N
S
+ CO 2
O
HB +
O
N
O
N
S
HO
O
HO
O
Breslow (1961) was the first to propose and provide evidence for the
C-2 carbanion, showing that this proton exhanges readily in D 2O
B
H
O
N
S
D
N
pH 5
S
CO 2
t1/2 ~ 2 min
N
S
ylide stabilized C
OH
O
HS
N
R
SH +
S
S
S
N
OH
S
OH
S
O
O
N
S
+
S
N
S
N
S
Acetyl CoA
Source of CH3CO 2H for
fatty acid biosynthesis
S
O
HO
Lipoic acid - source of 2e-, 2H+
2e-, 2H+
Elimination
-could also intercept with more pyruvate
O
O
Acyloin type condensation:
O Acetolactate synthetase
OH
SCoA
R
R
S
S
S
O
2. Oxidative decarboxylations:
exemplified by pyruvate dehydrogenase
BH +
N
S
BH +
HO H
N
HO
OH
HS
HS
OH
O
N
S
O
O
N
S +
O
O
OH
H
Redox Enzymes:
-NADPH/NADH
nicotinamide dependent (2e -, H +)
-FAD/FADPH flavin dependent 1e 1e - (2e - ,2 H +)
Note compounds with lower redox potential thoroughly reduce those with
higher redox potentials
# electrons
G=-nF E 0
Nicotinamide adenine dinucleotide (phosphate)
~23 kcal/mol
O
NH 2
O
N
O P
O
O
HO
O
O P O
2H + 2e- + 1/2O 2
Cu 2+ + 1e Cu+
+
riboflavin + 2H + 2eNAD + + 2H+ + 2e-
A
O
O
OH
HO
OH
O
HO H
O
O
O
NH 2
N
O
O
H H
O
O
NH 2
NH 2
Reactions of NAD + /NADPH+ Enzymes ( 2e- /H+)
O
H
OH
H
NH 2
O
NH 2
O
O
H
Overall Reaction:
O
reduce
N
N
NAD + + 2H+ +2e -
riboflavinH 2
NADH + H +
+0.81eV
+0.15eV
-0.22eV
-0.32eV
-use nicotinamide to reduce riboflavin
-molecular oxygen is reduced by riboflavin, not nicotinamide (1e - process)
BH +
H
H 2O
NADH + H +
E' 0= -0.32V
O
OH
O
H
OH
O + 2H+ +2e -
O
O
E' 0= -0.19V
O
OH
O
O
O
O
+ NADH + H +
H
Note that these are all sterospecific
Pro R
HR
O
E' 0=E'0(red) - E' 0(oxid) = -0.19V - (-0.32V) = 0.13V
N
To deduce whether it is pro R or pro S replace that
H by D and deterine R or S configuration
Si
Trasfers one
prochirial H
HS
O
Me
H
Pro S
NH 2
Zn2+
O
N
Re
O
NH 2
Me
O
HR H S
Flavin
OH
Reactions of Flavin Enzymes ( 2e- /2H+)
OH
O
10
N
10a
OH
N1 O
O
P
O
O
4a
5
N
4
O
NH
OH
Handle
N
H
OH
H
NH
NH 2
N
O
N
1e -
NH
N
O
NH
N
H
N
O
H+
N
NH
O
O
O
NADH
NAD +
O
1e - reduced
O
N
H
S
SH SH
1e -
N
O
A
O
O
H
O
N
oxidized
P
HO
2
N
O
S
OH
N
O
NH
OH
R
OH
R
O
O
O
2e- reduced
O
O
X
X
H
OH
OH
R
R
O
O
R
H
R
OH
Reaction of glutathione (glutathione reductase)
NADH +
Enz.Fl
+ GS-SG
2GSH +
Molecular oxygen
Enz.Fl
+
FAD
FADH 2
FAD
FAD
S
S
SH
SH
S
S
S
S
G
S
G S
G
S
NAD +
N
FAD
B
+ GSH
S
+ 2GSH
N
H
S
H
N
O
H+
N
N
NH
N
H
O
O
NH
O
O O
BH +
O OH
BH +
"peracid"
Source of +OH
N
N
N
H
N
O
NH
O
S
S
N
N
O
N
H
N
S
O
S
O
O
SH
BH +
OH
O
NH
N
H
NH
N
BH +
BH +
N
NH
N
O
O
OH
B
S
O
N
B
N
N
O
O
OH
O
NH
N
N
H
HO
N
O
+
NH
OH
HO