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: 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. 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). Basics: 1. Enantiomers: Chemically same substances but differ with respect to stereochemistry (also known as non-super-imposable mirror images). Example: Acetolactatic acid 2 Tautomerism: Special type of isomerism in which the isomers are easily inter-convertible at equilibrium. For acetolactic acid the equilibrium is at pH The enzyme ALDC does this at a much lower pH Diastereomers: Isomers with more than one chiral centre. There are 2 n 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. 2. Structural Activity Relationship (SAR): 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. Table 1 shows the different compounds synthesised Blue are known substrates or analogues Red are possible inhibitors 3. Synthesis: Numbers corresponds to the compound serial numbers in SAR table 4. Future work: 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. Figure 1. diagrammatic sketch of the enzymatic reaction of 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. Reference: 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, 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, 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, Acknowledgement: The Author would like to thank all his colleagues on 4 th 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. Figure 2 a and b taken from ref#1 show the predicted active site