Reaction of 1-Amino-2-methylenecyclopropane-1-carboxylate with 1-Aminocyclopropane-1-carboxylate Deaminase: Analysis and Mechanistic Implications ² Zongbao Zhao, ‡,§ Huawei Chen, § Keqiang Li, § Wensheng Du, § Shouming He, | and Hung-wen Liu* .‡,§ DiVision of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and Biochemistry, UniVersity of Texas, Austin, Texas 78712, Department of Chemistry, UniVersity of Minnesota, Minneapolis, Minnesota 55455, and Department of Chemistry, UniVersity of British Columbia, VancouVer, British Columbia, Canada ReceiVed September 4, 2002; ReVised Manuscript ReceiVed December 6, 2002 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 R-ketobutyric acid and ammonia. In an early study of this unusual C R -C  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 R-H from D-vinylglycine and may serve as the base to remove the -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 -H abstraction. Clearly, the cleavage of ACC to R-ketobutyrate by ACC deaminase represents an intriguing conversion beyond the common scope entailed by coenzyme B 6 dependent catalysts. 1-Aminocyclopropane-1-carboxylate (ACC) 1 deaminase, originally isolated from Pseudomonas grown on ACC (1) as the sole nitrogen source, is a pyridoxal 5′-phosphate (PLP) linked catalyst with a unique capacity to break the cyclo- propane ring of ACC to give R-ketobutyric acid (2) and ammonia (Scheme 1) (1). Although the catalytic roles of PLP are amazingly versatile, the common theme among the various catalytic transformations of PLP-dependent enzymes is the ability of this cofactor to act as an electron sink, ² This work was supported in part by a National Institutes of Health grant (GM40541). K.L. was a recipient of a National Research Service Award (NIGMS GM17412). * To whom correspondence should be addressed. Fax: 512-471- 2746. E-mail: h.w.liu@mail.utexas.edu. ‡ University of Texas. § University of Minnesota. | University of British Columbia. 1 Abbreviations: ACC, 1-aminocyclopropane-1-carboxylic acid; DTNB, 5,5′-dithiobis(2-nitrobenzoate); 2-methylene-ACC, 1-amino- 2-methylenecyclopropane-1-carboxylic acid; DEAE, diethylaminoethyl; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; IPTG, isopropyl thio--galactoside; LB, Luria-Bertani; PCR, polymerase chain reaction; PLP, pyridoxal 5′-phosphate; PMSF, phenylmethane- sulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TCPK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; TMS, tetramethylsilane; WT, wild type. Scheme 1 2089 Biochemistry 2003, 42, 2089-2103 10.1021/bi020567n CCC: $25.00 © 2003 American Chemical Society Published on Web 01/29/2003