Probing chemical mechanism of two enzyme-catalyzed reactions by chiral substrate analogues

Enzymes are biological catalysts which greatly accelerate the rate of chemical reactions with remarkable substrate specificity and stereoselectivity. To optimize their catalytic abilities, many enzymes have also evolved to cooperate with coenzymes. The variety of coenzymes largely enhances the scope of enzyme-catalyzed reactions that are not accessible to the twenty canonical amino acids. In general, the chiral enzyme active sites allow enzymes to selectively bind substrates with a specific conformation which can lower the activation energy of the reactions by stabilizing high energy intermediates. Therefore, if the substrate of an enzyme contains chirality, in many cases, enzymes preferably accept one of the stereoisomers for catalysis. Inspired by this phenomenon, the work described in this dissertation focuses on mechanistic studies of two enzyme-catalyzed unusual chemical reactions by using chemically synthesized chiral substrate analogues. The first section of this dissertation focuses on 1-aminocyclopropane-1-carboxylic acid deaminase (ACCD), an enzyme that plays a role in regulating the production of the potent plant hormone, ethylene. ACCD is a pyridoxal-5ʹ-phosphate (PLP)-dependent enzyme that catalyzes a C-C bond cleavage event that is unique among the PLP-dependent enzymes. (R)- and (S)-2,2-difluoro-1-aminocyclopropane-1-carboxylic acid are synthesized as mechanism based inhibitors. Our studies suggest that the previously proposed acid-catalyzed mechanism for the ACCD-catalyzed reaction is less likely, and instead, a nucleophilic attack mechanism is consistent with the accumulated experimental results from the mechanistic studies of the ACCD reaction. The second section of this dissertation focuses on (E)-4-hydroxyl-3-methylbutenyl-1-diphosphate reductase (IspH), an essential enzyme in the non-mevalonate pathway (MEP pathway) for isoprenoid biosynthesis that employs a [4Fe-4S] cluster as cofactor for catalysis. Stereospecific labeling of tritium on the substrate and substrate analogue of IspH are used to generate chiral methyl groups in the products of the IspH reaction via catalysis in D2O-based buffer condition. Our results suggest that a configuration inversion at C4 of the substrate during the catalysis of IspH reaction is a universal step among all currently proposed mechanisms for the IspH reaction. Furthermore, we also provide the first experimental evidence for the direction of protonation of the allyl anion intermediate during IspH catalysis.