Mco
A
Studying the Mechanism of Acetolactate Decarboxylase (ALDC)
Amit Anand, Martin Wills
Department of Chemistry, University of Warwick, UK.
Project One: Synthesis of Substrate, Substrate Analogues and Inhibitors
1.Introduction:
Basics:
Acetolactate decarboxylase or ALDC is an enzyme which decarboxylates the natural substrate
Acetolactate to Acetoin. Moreover it also converts the R enantiomer to S enantiomer before
decarboxylating it.
1. Enantiomers:
H3C
3
C
2 HOOC
H3C
4
O
H3C
CH3
O
C
ALDC
2C
ALDC
4
3
(S) C
COOH 2
1 HO
CH3
C (R)
OH 1
Chemically same substances but differ with respect to stereochemistry
(also known as non-super-imposable mirror images).
H3C
Example: Acetolactatic acid
CH3
3
4 H
O
C (R)
OH
H3C
3
acetoin
Acetolactate
This enzyme has been used in the brewing industry for more than a decade. It reduces the
maturation time of around 10 weeks to 24 hours. The real mechanism is unknown. Recently a
successful X-ray crystal structure of the enzyme has been reported. (ref#1).
2. Structural Activity Relationship (SAR):
O
O
C
C
3
2 HOOC
(S)
C (R)
C
COOH 2
OH 1
1 HO
H3C
4CH3
4
2-Hydroxy-2-methyl-3-oxo-butyric acid
(Acetolactate)
1
2 Tautomerism:
Special type of isomerism in which the isomers are easily inter-convertible
at equilibrium. For acetolactic acid the equilibrium is at pH 12-13. The enzyme ALDC does this
at a much lower pH 6-7.
Acetolactate
O
1
OH
C
H3C 3(R) C CH
34
Based on the structure of the natural substrate (S)-acetolactic acid, some analogues and
inhibitors were synthesised as shown in the table 1 below. 1R, 2a and 2b are known analogues
where as the rest of the compounds are expected inhibitors as they lack one or more functional
groups present in the natural substrate.
H
OH
C
1 HO
O
C (S)
HOOC
1C
2
H3C
C
H3C
COOH
2
OOC
3
CH3
CH3
O
Transition state
Table 1 shows the different compounds synthesised
Natural Substrate:(S) Acetolactate
3
2
O
HO
2-Hydroxy-2-methyl-3-oxobutyric acid
Red are
possible
inhibitors
C
C
4 3HC
Blue are known
substrates or
analogues
3 Diastereomers: Isomers with more than one chiral centre. There are 2n potential
diastereomers, where n is the number of chiral centres. Dihydroxy inhibitor compounds
proposed in the SAR section have two chiral centres. Hence they will have four diastereomers.
Making diastereomer inhibitors will help us in identifying the type of chiral molecule that the
enzyme prefers.
CH3
COOH
1
5
Funtional groups
1
Compound Serial Numbers
-CH3
1S -CH3
1
2
4
5
-OH
-CH3
-COOH
=O
-COOH
1R -CH3
=O
=O
=O
-OH
2a -CH3
=O
-OH
2b
3
-C2H5
-CH3
=O
=O
IUPAC Names
3
-OH
-OH
-COOH
2-Hydroxy-2-methyl-3-oxo-butyric acid
-COOH
-CH3
-COOCH3
2,3-Dihydroxy-2-methyl-butyric acid
methyl ester
-OH
-CH3
-COOH
2,3-Dihydroxy-2-methyl-butyric acid
=O
-OH
-C2H5
-COOC2H5
2-Ethyl-2-hydroxy-3-oxo-butyric acid
ethyl ester
-OH
-OH
-C2H5
-CH3
5
-CH3
-OH
6
-CH3
7
-CH3
8
-CH3
-OH
-OH
-COOC2H5
-C2H5 -COOH
COOX
X OH
(R) C
OH
(S)
C (R)
OH C X
CH3
C (R)
H
H CH3
X
C (S)
H
H
CH3
C
(R)
CH3
C (S)
CH3
C (R)
HO
HO
CH3
H
H
2,3-Dihydroxy-2-methyl-butyric acid
C CH3
(R)
C CH
3
(S)
H
Characterising the active site by taking X-ray snap shot of a transition state between the
substrate and the enzyme. Other choices apart from substrate is substrate’s ethyl analogue
and Inhibitors.
OH COOX
OH
CH3
OH
OH
2-Ethyl-2,3-dihydroxy-butyric acid
When X is -CH3 then the compound is (3)
When X is -C2H5 then the compound is (6)
X
X
Pig Liver
X
HOOC C OH
XOOC C OH Esterase
CH3
KMnO4/AcOH/Acetone/H2O
C
C
C C CO2X
O
O
[O]
H
C
3
H3C
H
When X is -CH3 then the
compound is (1)
[H]
When X is -C2H5 then the
NaBH4
compound is (2)
When X is -CH3 then the compound is (4)
When X is -C2H5 then the compound is (7)
(S)
C
CH3
(R) C
OH
COOH
4. Future work:
3. Synthesis:
OH
C (S)
COOH
2-Ethyl-2,3-dihydroxy-butyric acid
ethyl ester
Numbers corresponds to the
compound serial numbers in
SAR table
COOX
OH
2-Hydroxy-methyl-oxo-butyric acid
methyl ester
-COOCH3
-OH
(5)
(S)
C
3-Ethyl-3-hydroxy-2-oxo-butyric acid
-COOH
-CH3
OH
2-Hydroxy-2-methyl-3-oxo-butyric acid
OH
-C2H5
COOH
COOH
-CH3
4
-OH
-CH3
2-Hydroxy-2-methyl-3-oxo-butyric acid
CH3
Figure 1. diagrammatic sketch of the enzymatic reaction of ALDC
CO2-
CO2-
O
(S) C OH
O
+
C CH
3
CH3
3
CH3
Acetolactic acid
(S) C OH
C CH
ALDC
Transition State
H
O
(R) C OH
+ CO2 +
C
CH3
CH3
Acetoin
ALDC
Proposed Active Site: From a recent X-ray crystal structure of the enzyme, a zinc metal ion
binding site was proposed to be the active site. The structure shows, the zinc with three
histidines, two water molecules and a glutamate (see Figure 2a). As shown in figure 2b
Glutamate Glu 93 and Arginine Arg 173 which interact with the zinc ion via water molecules
are likely to play a key role in catalysis. There is also an absolutely conserved Threonine Thr
86 nearby which may play an important role of holding the carbon dioxide leaving group.
COOX
OH
(R) C X
OH
C CH
3
(S)
H
Figure 2 a and b taken from
ref#1 show the predicted
active site
LiOH/Isopropanol/H2o
Ester hydrolysis
Reference:
When X is -CH3 then the compound is (5)
When X is -C2H5 then the compound is (8)
OH
COOH
(R) C
OH
COOH
OH
OH
H
H
C(S)
C (S)
CH3
C (R)
X
COOH
X
OH
C (S)
X
C (R)
H CH3
CH3 OH
COOH
OH
OH
H
C X
(R)
C CH
3
(S)
Ref#1 S. Najmudin, J. T. Andersen, S. A. Patkar, T. V. Borchert, D. H. G. Crout and V. Fülöp Purification, crystallization and preliminary X-ray
crystallographic studies on acetolactate decarboxylase Acta Cryst. (2003). D59, 1073-1075
Ref#2 David H. G. Crout, C. Rupert McIntyre, Nathaniel W. Alcock, Stereoelectronic control of the tertiary ketol rearrangement: implications for the
mechanism of the reaction catalysed by the enzymes of branched-chain amino acid metabolism, reductoisomerase and
acetolactate decarboxylase J. Chem. Soc., Perkin Trans. 2, 1991, 53-62
Ref#3 David H. G. Crout, Edward R. Lee, David P. J. Pearson, Stereoelectronic control of the base-catalysed rearrangement of 2-hydroxy 3-oxo
carboxylates J. Chem. Soc., Perkin Trans. 2, 1991, 381-385
Acknowledgement:
The Author would like to thank all his colleagues on 4th floor Chemistry Building and MOAC students for all the support. A special thanks to Prof. Martin
Wills and Prof. Alison Rodger for giving him an opportunity to work in a multidisciplinary environment.