| Abstract
| - Glutamate 47 is conserved in 1-aminocyclopropane-1-carboxylate (ACC) synthases and ispositioned near the sulfonium pole of (S,S)-S-adenosyl-l-methionine (SAM) in the modeled pyridoxalphosphate quinonoid complex with SAM. E47Q and E47D constructs of ACC synthase were made toinvestigate a putative ionic interaction between Glu47 and SAM. The kcat/Km values for the conversion of(S,S)-SAM to ACC and methylthioadenosine (MTA) are depressed 630- and 25-fold for the E47Q andE47D enzymes, respectively. The decreases in the specificity constants are due to reductions in kcat forboth mutant enzymes, and a 5-fold increase in Km for the E47Q enzyme. Importantly, much smallereffects were observed for the kinetic parameters of reactions with the alternate substrates l-vinylglycine(l-VG) (deamination to form α-ketobutyrate and ammonia) and l-alanine (transamination to form pyruvate),which have uncharged side chains. l-VG is both a substrate and a mechanism-based inactivator of theenzyme [Feng, L., and Kirsch, J. F. (2000) Biochemistry 39, 2436−2444], but the partition ratio, kcat/kinact, is unaffected by the Glu47 mutations. ACC synthase primarily catalyzes the β,γ-elimination ofMTA from the (R,S) diastereomer of SAM to produce l-VG [Satoh, S., and Yang, S. F. (1989) Arch.Biochem. Biophys. 271, 107−112], but catalyzes the formation of ACC to a lesser extent via α,γ-eliminationof MTA. The partition ratios for (α,γ/β,γ)-elimination on (R,S)-SAM are 0.4, ≤0.014, and ≤0.08 for thewild-type, E47Q, and E47D enzymes, respectively. The results of these experiments strongly support arole for Glu47 as an anchor for the sulfonium pole of (S,S)-SAM, and consequently a role as an activesite determinant of reaction specificity.
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