Download Mechanism of Carbanion Stabilization by PLP, Cont`d

King Saud University
College of Science
Department of
Biochemistry
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Part 3
Coenzymes-Dependent Enzyme
Mechanism
Professor A. S. Alhomida
1
2
Mechanism of Carbanion
Stabilization by PLP
3
Mechanism of Carbanion
Stabilization by PLP
H
R
Lys
P
O
O
HO
Internal aldimine
(PLP-Enz Schiff base)
C
P
a
CO2
NH
O
CH
O
b
H
N
O
O
Enz
C
H
CH
O
O
HO
N
H
H
R
N
H
CH3
Lys
H
C
C
H
NH3
NH2
Enz
External aldimine
(PLP-substrate Schiff base)
CO2
a-Amino Acid
CH3
4
Mechanism of Carbanion
Stabilization by PLP, Cont’d
H
R
O
O
P
B:
H
b
a
C
C
H
NH
H
R
CO2
O
CH
O
O
HO
O
P
BH
b
a
C
C
H
NH
CO2
CH
O
O
HO
N
H
CH3
N
H
CH3
I
Stabilized carbanion resonance
5
Mechanism of Carbanion
Stabilization by PLP, Cont’d
H
H
b a
R
O
O
P
C
C
H
NH
BH
CO2
R
O
CH
O
O
HO
O
P
HO
N
H
II
CH3
b a
C
C
H
NH
BH
CO2
CH
O
O
..
N
H
CH3
III
Stabilized carbanion resonance
6
Mechanism of Carbanion
Stabilization by PLP, Cont’d
H
R
C
H
b a
C
b
CO2
R
C
a
CO2
C
BH
H
O
O
P
B:
NH
H
O
CH
O
O
HO
O
P
H
NH
C H
O
O
HO
N
H
CH3
III
N
H
CH3
IV
Stabilized carbanion resonance
7
Mechanism of Carbanion
Stabilization by PLP, Cont’d
H
R
C
BH
H
O
O
P
H
b
a
C
R
CO2
BH
NH
H
O
C H
O
C
O
O
P
a
CO2
C
NH
C H
O
HO
HO
N
H
b
CH3
V
O
..
N
H
CH3
VI
Stabilized carbanion resonance
8
Mechanism of Carbanion
Stabilization by PLP, Cont’d
H
R
C
b
H
a
C
b a
CO2
R
C
C
H
NH
CO2
BH
H
O
O
P
NH
C H
O
O
HO
O
O
BH
P
O
HO
N
H
CH3
VI
C H
O
..
N
H
CH3
III
Stabilized carbanion resonance
9
Mechanism of Carbanion
Stabilization by PLP, Cont’d
B
BH
H
H
R
O
P
b
ba
R
CO2
C
C
H
NH
O
HO
For determination
of stereochemistry
of amino acid
formed
O
..
N
H
O
P
a
C
C
H
NH
O
C H
O
H
CO2
C H
O
O
HO
CH3
N
H
CH3
VII
III
Stabilized carbanion resonance
10
Mechanism of Carbanion
Stabilization by PLP, Cont’d
H
B:
R
O
O
P
b a
CO2
C
C
H
NH
H
R
O
C H
O
b
BH
O
HO
O
P
a
C
C
H
NH
H
CO2
C H
O
O
etc
HO
N
H
II
CH3
N
H
CH3
VIII
Stabilized carbanion resonance
11
Jencks’ Statement
• The versatile chemistry of pyridoxal
phosphate offers a rich learning experience
for the student of mechanistic chemistry
• Professor W. Jencks, in his classic text,
Catalysis in Chemistry and Enzymology,
writes:
– “It has been said that God created an organism especially
adapted to help the biologist find an answer to every
question about the physiology of living systems;
12
Jencks’ Statement, Cont’d
– if this is so it must be concluded that pyridoxal
phosphate was created to provide satisfaction and
enlightenment to those enzymologists and
chemists who enjoy pushing electrons, for no
other coenzyme is involved in such a wide variety
of reactions, in both enzyme and model systems,
which can be reasonably interpreted in terms of
the chemical properties of the coenzyme
13
Jencks’ Statement, Cont’d
– Most of these reactions are made possible by a
common structural feature
– That is, electron withdrawal toward the cationic
nitrogen atom of the imine and into the electron
sink of the pyridoxal ring from the a carbon atom
of the attached amino acid activates all three of
the substituents on this carbon atom for reactions
which require electron withdrawal from this atom”*
*Jencks,
William P., 1969. Catalysis in Chemistry and
Enzymology. New York: McGraw-Hill
14
Biochemical Functions of
Pyridoxal phosphate
1.
2.
3.
4.
5.
6.
7.
Decarboxylation of amino acids
Transaminase reactions
Racemization reactions
Aldol cleavage reactions
Transulfuration reactions
Conversion of tryptophan to niacin
Conversion of linoleic acid into arachidonic
acid (prostaglandin precursor)
8. Formation of sphingolipids
15
Transamination Reactions
16
• The term transamination refers to the
interconversion of carbonyl and amino
groups. Condensation of an amine with an
aldehyde as shown below gives an imine.
What is formally only a tautomerisation
reaction converts imine A into its tautomer B
which upon hydrolysis yields the
"transaminated" products. i.e. the product in
which the amine and the carbonyl group have
been swapped..
17
• This is a highly simplified view of the
transamination reaction.
• Firstly, aldehydes do not occur in
biological systems due to their chemical
instability. The biological equivalent of
aldehydes are imines.
18
• Firstly, aldehydes do not occur in
biological systems due to their chemical
instability. The biological equivalent of
aldehydes are imines.
19
• Secondly, imines are chemically stable
towards this type of tautomerisation reaction.
An enzyme is required to effect this
transformation and the enzymes employs a
co-factor (or prosthetic group).This cofactor is
pyridoxal phosphate (PLP).
20
• PLP is attached to the enzyme forms an
imine with a lysine residue. This link attaches
the co-factor to the enzyme and converts the
aldehyde into its biological equivalent, the
imine.
• The conversion of amino acids into a-keto
acids (also sometimes referred to as a-oxoacids) is a central reaction of primary and
secondary metabolism.
21
• In the first step of
transamination
reactions,
pyridoxalphosphate
in its biological form
of imine is tranferred
to the substrate
amino acid.
22
23
• Then the PLP-dependent enzymes
catalyses the tautomerisation of the
imime.
24
• In the final step, hydrolysis of the imine gives the products.
• Note, that pyridoxal phosphate (PLP) has been converted into
pyridoxamine by the transamination reaction. A second
transamination step is required to convert pyridoxamine back
into PLP. This restores the co-factor and the enzyme can carry
out another transamination reaction.
25
Mechanism of PLP-catalysed
transaminations
• The a-hydrogen of the imine is in
conjugation with the protonated
pyridinium nitrogen. The positively
chareged nitrogen increases the aciditiy
of the a-hydrogen and facilitates proton
abstraction. The product is an extended
conjugated system incorporating both
an imine and an enamine.
26
27
• In the final step, protonation occurs at
the d-carbon to the pyridine nitrogen,
thus restoring the aromatic system.
Hydrolysis of the imine gives the final
products.
28
Decarboxylation and PLP
• Decarboxylation reactions are important
in biological systems because
intermediates which are chemically
disposed for decarboxylation, such as
b-keto acids, occur frequently in primary
and secondary metabolism.
29
• a-Keto acids are chemically not predisposed
towards decarboxylation. This is reflected in
much higher temperatures required to effect
the above transformation. Nature uses
enzymes for this reaction which carry PLP as
co-factor. The schemes below shows the
decarboxylation of an a-amino acid.
30
• The amino acid is bound to to PLP as
the imine in the first step.
31
• In the actual decarboxylation step, the
electronic effects are the same: the
pyridine nitrogen acts as an electronwithdrawing group, this time facilitating
deprotonation of the carboxylic acid
group. Loss of carbondioxide and
hydrolysis of the imine gives the
reaction products.
32
Transamination Reactions
a-Amino acid
a-Keto acid
a-Keto acid
a-Amino acid
33
Transamination Reactions
• Most common amino acids can be converted
into the corresponding keto acid by
transamination
• This reaction swaps the amino group from
one amino acid to a different keto acid,
thereby generating a new pairing of amino
acid and keto acid
• There is no overall loss or gain of nitrogen
from the system
34
Transamination Reactions, Cont’d
• Transamination reactions are readily
reversible, and the equilibrium constant is
close to 1
• One of the two substrate pairs is usually Glu
and its corresponding keto acid a-KG
35
Transamination Reactions, Cont’d
• The effect of transamination reactions is to
collect the amino groups from many different
amino acids in the form of L-Glu
• The Glu then functions as an amino group
donor for biosynthetic pathways or for
excretion pathways that lead to the
elimination of nitrogenous waste products
36
Transamination Reactions, Cont’d
• The substrates bind to the enzyme active
center one at a time, and the function of the
pyridoxal phosphate is to act as a temporary
store of amino groups until the next substrate
comes along
• In the process the pyridoxal phosphate is
converted into pyridoxamine phosphate, and
then back again Enzymologists call this a
ping pong mechanism
37
Transamination Reactions, Cont’d
• The condensation between the a-amino
group and the aromatic aldehyde to form a
Schiff base makes the a-carbon atom
chemically reactive, so the isomerization of
the Schiff base takes place very easily
• Many of the enyzmes that metabolize amino
acids require PLP as a cofactor
• Unexpectedly, this compound also serves in a
completely different manner in the active
center of glycogen phosphorylase
38
• Comparison of the
active sites of Laspartate
aminotransferase
(left) and D-amino
acid
aminotransferase
(right)
39
• The three-dimensional
structures of bacterial
D-amino acid
aminotransferase (top)
and human
mitochondrial branchedchain L-amino acid
aminotransferase
(bottom)
40
Aspartate Transaminase
(Aspartate Aminotransferase)
41
Aspartate Transaminase
• Aspartate transaminase (AST) also called
serum glutamic oxaloacetic transaminase
(SGOT) or aspartate aminotransferase
(ASAT/AAT) (EC 2.6.1.1) is similar to alanine
transaminase (ALT) in that it is another
enzyme associated with liver parenchymal
cells
• PLP coenzyme provides an aldehyde group
to the enzyme, which is not available among
the side chains of the 20 amino acids found in
proteins
42
Aspartate Transaminase, Cont’d
• The phosphate group provides a way to bind
the coenzyme to the enzyme via a strong
ionic interaction
• The aldehyde group readily reacts with
primary amines like the a-amino groups of
amino acids
• This process activates the amino group so
that it can be cleaved by water
43
Aspartate Transaminase, Cont’d
• This releases the keto-acid core of the amino
acid and leaves the amino group on the
enzyme
• Now the acceptor keto-acid binds and reacts
with the activated amino group to form the
new amino acid
44
Aspartate Transaminase, Cont’d
• The mitochondrial aspartate transaminase
provides an especially well studied example
of PLP as a coenzyme for the transamination
reactions
• The results of X-ray crystallographic studies
provided detailed views of how PLP and
substrates are bound and confirmed much of
the proposed catalytic mechanism
45
Aspartate Transaminase, Cont’d
• The enzyme is a dimer if identical subunits
and it consists of a large domain and a small
one
• PLP is bound to the large domain, in a pocket
near the subunit interface
• In the absence of substrate, the aldehyde
group of PLP is in a Schiff base linkage with
Lys-258
• Arg-386 interacts with the a-carboxylate
group of the substrate, helping to orient the
substrate appropriately in the active site 46
Structure of Aspartate
Transaminase
• The active site of
enzyme includes PLP
attached to the enzyme
by Schiff base linkage
with Lys-258
• Arg-386 residue in the
active site helps orient
substrates by binding to
their a-carboxylate
groups
47
Structure of Aspartate
Transaminase
• Schematic diagram of the
active site of E. coli
aspartate aminotransferase
• Substrate specificity for the
negatively charged aspartic
acid substrate is determined
by the positively charged
guanidino groups of Arg-386
and Arg-292, which have no
catalytic role
• Mutation of Arg-292 to Asp
produces an enzyme that
prefers Arg to Asp as a
substrate
48
Stereochemistry of Aspartate
Transaminase Reaction
• PLP enzymes cleave one of
three bonds at the Ca atom
of amino acids
• For example, bond a is
cleaved by
aminotransferase, bond b by
dehydrogenase, and bond c
by aldolase
• How can the same amino
acid-PLP Schiff base be
involved in the cleavage of
the different bonds to an
amino acid Ca?
49
Stereochemistry of Aspartate
Transaminase Reaction, Cont’d
• For electrons to be withdrawn into the
conjugated ring system of PLP, the p-orbital
system of PLP must overlap with the bonding
orbital containing the electron pair being
delocalized
• This is possible only if the bond being broken
lies in the plane perpendicular to the plane of
the PLP p-orbital system
• Different bonds to Ca can be placed in this
plane by rotation about the Ca-N bond
50
Stereochemistry of Aspartate
Transaminase Reaction, Cont’d
• Each enzyme specifically cleaves its
corresponding bond because the enzyme
binds the amino acid-PLP Schiff base adduct
with this bond in the plane perpendicular to
that of the PLP ring
• This is an example of stereoelectronic
assistance (effect)
• The enzyme binds substrate in a
conformation that minimizes the electronic
energy of the transition state
51
Stereochemistry of Aspartate
Transaminase Reaction, Cont’d
• Bond orientation in a
PLP–amino acid Schiff
base
• The p-orbital framework
of a PLP–amino acid
Schiff base
• The bond to Ca in the
plane perpendicular to
the PLP p-orbital
system
52
Stereochemistry of Aspartate
Transaminase Reaction, Cont’d
• In PLP-dependent
transaminase’s active site,
the addition of H+ from Lys
residue to the bottom face of
the quinoid intermediate
determines the Lconfiguration of the amino
acid product
• The conserved Arg residue
interacts with the acarboxylate group and helps
establish the appropriate
geometry of the quinonid
intermediate
53
Mechanism of L-Configuration of
Amino Acids Produced
B
BH
H
H
R
O
P
b
ba
R
CO2
C
C
H
NH
O
HO
For determination
of stereochemistry
of amino acid
formed
O
..
N
H
O
P
a
C
C
H
NH
O
C H
O
H
CO2
C H
O
O
HO
CH3
N
H
CH3
VII
III
Stabilized carbanion resonance
54
Stereochemistry of Aspartate
Transaminase Reaction, Cont’d
• The orientation about
the NH-Ca bond
determines the most
favored reaction
catalyzed by PLPdependent enzymes
• The bond that is most
nearly perpendicular to
the p orbital of the PLP
electron sink is most
easily cleaved
55
Stereochemistry of Aspartate
Transaminase Reaction, Cont’d
• In PLP-dependent
transaminases, Ca-H
bond is most nearly
perpendicular to the p
orbital system and is
cleaved
• In SHMT, a small
rotation about N-Ca
bond places the Ca-Cb
bond perpendicular to
the p system, favoring
its cleavage
56
Mechanism of Aspartate
Transaminase
57
Reaction of Aspartate
Transaminase
H
Asp Transaminase
CO2
O2C
L-Asp
CO2
O2C
O
NH3
PLP
PMP
OAA
NH3
H
O2C
CO2
CO2
O2C
NH3
L-Glu
O
a-KG
58
Reaction of Aspartate
Transaminase
OAA
L-Asp
E-PLP
PLP-Asp
PLP-OAA
E-PMP
L-Glu
a-KG
PLP-a-KG
PLP-Glu
Ping Pong Mechanism
59
E-PLP
Active Site of Asp Transaminase
Lys-258
N
Arg-292
H2N
Arg-386
H2
NH2
O
O
Both carboxylate
groups of Asp are
bound by
electrostatic
interactions to the
active site Arg-292
and Arg-386
General base
H2N
NH2
O
H
O
NH3
H
O
O P O
O
O
N
CH3
H
External aldimine (PLP-Asp Schiff base)
60
Mechanism of Asp Transaminase
Lys
BH+
Lys
N
H2
O
O P O
O
C H
O
N
O
ENZ
O
H
O
O P O
O
C
N
H
H
O
B:
ENZ
CH3
H
N
H
CH3
Tetrahedral intermediate
PLP
61
Mechanism of Asp Transaminase,
Cont’d
Lys
H
N
O
Asp
H2O
O P O
O
C
H
O
ENZ
H
N
CH3
H
PLP-Enzyme Schiff base
B:
(Enzyme aldimine)
H
H N C COO
H
CH2
COO
Asp
62
Mechanism of Asp Transaminase,
Cont’d
Lys
H
H
N
O
O P O
O
H
..
N C
C
O
N
H
COO
H CH2
CH3
COO
H
Tetrahedral intermediate
63
Mechanism of Asp Transaminase,
Cont’d
Abstract a-carbon
:N
Lys
H2
COO
H
HN
C H CH2
O
O
P
O
C
H2C
COO
N
CH3
N
H3
CH
O
O
P
O
Lys
COO
NH
COO
O
O
C
O
O
..
N
CH3
H
H
PLP-Asp Schiff base
(Asp aldimine)
Quinonoid
64
Mechanism of Asp Transaminase,
Cont’d
H
COO
O
COO
H2 C
C
N
H2
NH
O
H2O
O P
O
H
C
Lys
COO
O
CH3
H
Kitimine
C
H
C
O
H
H
N
H2C
O P
O
B:
Lys
COO
N
H2
NH
H
O
O
BH+
B:
H
O
O
O
N
H
CH3
a -KG
Tetrahedral intermediate
C
COO
H2C
COO
OAA
65
Mechanism of Asp Transaminase,
Cont’d
Lys
..
N
H2
NH2
O
O P
O
H
C
COO
C
N
O
CH3
H
PMP
H2 C
BH+
H
O
O
H2C
2
COO
a-KG
Lys
COO
BH+
O
O P
O
N
H2
2
HO
C
COO
H
N
H
H
C
H
B:
O
O
N
H2O
CH3
H
Tetrahedral intermediate
66
Mechanism of Asp Transaminase,
Cont’d
Lys
COO
Protonation
at a-carbon H2C 2
C
B:
O
O P
O
H
N
H
C
H
N
H
N
H2C
H
COO
H
BH+
H
C
H
N
C
O
O
O
O P
N
CH3
H
Kitimine
Lys
COO
O
2
H
COO
H
O
O
N
CH3
H
Glu aldimine
67
H
Mechanism of Asp Transaminase,
Cont’d
Lys
COO
H2C
BH
+
H
O
O P
O
C
H
N
H
C
B:
2
Lys
O
O P
O
C
H
O
O
N
CH3
BH+
H
H
Enzyme aldimine
B:
N
H
O
COO
H
H
N
O
N
COO
CH3
(PLP-Enzyme Schiff base)
H
H2C
Tetrahedral intermediate
2
H
C
COO
H
N
H
H
Glu
68
Experimental Evidences for the
Role of Lys-258, Arg-385 and Arg292
• By using site-directed mutagenesis
techniques by replacing Lys-258 for Ala gives
a completely inactive mutant enzyme
• Replacing Lys-258 for Cys, the mutant
enzyme is similarly inactive, however, if this
enzyme is alkylated with 2-bromoethylalanine
an active enzyme is obtained which contains
a thioether analog of Lys at the active site
69
Experimental Evidences for the
Role of Lys-258, Arg-385 and Arg292, Cont’d
• This enzyme has 7% of the activity of wildtype enzyme with a slightly shifted pH rate
profile of enzymatic activity
• Since the thioether-containing Lys analog is
slightly less basic than Lys
• By replacing Arg-292 by other amino acids,
mutation of Arg-292 to Asp-292 gave an
enzyme whose catalytic efficiency for L-Asp
has dropped from 34500 to 0.07 M-1s-1
70
Experimental Evidences for the
Role of Lys-258, Arg-385 and Arg292, Cont’d
• However, mutant enzyme was found to
be capable of processing L-amino acid
substrates containing positively charged
side chains (Arg, Lys, and ornithine)
which would interact favorably with Asp292 with kcat/Km of 0.43 M-1s-1
71