Glutamate 47 in 1-aminocyclopropane-1-carboxylate synthase is a major specificity determinant

Glutamate 47 is conserved in 1-aminocyclopropane-1-carboxylate (ACC) syntha ses and is positioned near the sulfonium pole of (S,S)-S-adenosyl-L-methion ine (SAM) in the modeled pyridoxal phosphate quinonoid complex with SAM. E4 7Q and E47D constructs of ACC synthase were made to investigate a putative ionic interaction between Glu47 and SAM. The k(cat)/K-m values for the conv ersion of (S,S)-SAM to ACC and methylthioadenosine (MTA) are depressed 630- and 25-fold for the E47Q and E47D enzymes, respectively. The decreases in the specificity constants are due to reductions in k(cat) for both mutant e nzymes, and a 5-fold increase in K-m for the E47Q enzyme. Importantly, much smaller effects were observed for the kinetic parameters of reactions with the alternate substrates L-vinylolycine (L-VG) (deamination to form alpha -ketobutyrate and ammonia) and L-alanine (transamination to form pyruvate), which have uncharged side chains. LNG is both a substrate and a mechanism- based inactivator of the enzyme [Feng, L., and Kirsch, J. F. (2000) Biochem istry 39, 2436-2444], but the partition ratio, k(cat)/k(inact), is unaffect ed by the Glu47 mutations. ACC synthase primarily catalyzes the beta,gamma -elimination of MTA from the (R,S) diastereomer of SAM to produce LNG [Sato h, S., and Yang, S. F. (1989) Arch. Biochem. Biophys. 271, 107-112], but ca talyzes the formation of ACC to a lesser extent via alpha,gamma -eliminatio n of MTA. The partition ratios for (alpha,gamma/beta,gamma)-elimination on (R,S)-SAM are 0.4, less than or equal to0.014, and less than or equal to0.0 8 for the wild-type, E47Q, and E47D enzymes, respectively. The results of t hese experiments strongly support a role for Glu47 as an anchor for the sul fonium pole of (S,S)-SAM, and consequently a role as an active site determi nant of reaction specificity.