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  • Title: Reaction of 1-amino-2-methylenecyclopropane-1-carboxylate with 1-aminocyclopropane-1-carboxylate deaminase: analysis and mechanistic implications.
    Author: Zhao Z, Chen H, Li K, Du W, He S, Liu HW.
    Journal: Biochemistry; 2003 Feb 25; 42(7):2089-103. PubMed ID: 12590597.
    Abstract:
    1-aminocyclopropane-1-carboxylate (ACC) deaminase is a pyridoxal 5'-phosphate (PLP) dependent enzyme which catalyzes the opening of the cyclopropane ring of ACC to give alpha-ketobutyric acid and ammonia. In an early study of this unusual C(alpha)-C(beta) ring cleavage reaction, 1-amino-2-methylenecyclopropane-1-carboxylic acid (2-methylene-ACC) was shown to be an irreversible inhibitor of ACC deaminase. The sole turnover product was identified as 3-methyl-2-oxobutenoic acid. These results provided strong evidence supporting the ring cleavage of ACC via a nucleophilic addition initiated process, thus establishing an unprecedented mechanism of coenzyme B(6) dependent catalysis. To gain further insight into this inactivation, tritiated 2-methylene-ACC was prepared and used to trap the critical enzyme nucleophiles. Our results revealed that inactivation resulted in the modification of an active site residue, Ser-78. However, an additional 5 equiv of inhibitor was also found to be incorporated into the inactivated enzyme after prolonged incubation. In addition to Ser-78, other nucleophilic residues modified include Lys-26, Cys-41, Cys-162, and Lys-245. The location of the remaining unidentified nucleophile has been narrowed down to be one of the residues between 150 and 180. Labeling at sites outside of the active site is not enzyme catalyzed and may be a consequence of the inherent reactivity of 2-methylene-ACC. Further experiments showed that Ser-78 is responsible for abstracting the alpha-H from d-vinylglycine and may serve as the base to remove the beta-H in the catalysis of ACC. However, it is also likely that Ser-78 serves as the active site nucleophile that attacks the cyclopropane ring and initiates the fragmentation of ACC, while the conserved Lys-51 is the base required for beta-H abstraction. Clearly, the cleavage of ACC to alpha-ketobutyrate by ACC deaminase represents an intriguing conversion beyond the common scope entailed by coenzyme B(6) dependent catalysts.
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