Glutamate 47 in 1-aminocyclopropane-1-carboxylate synthase is a major specificity determinant.
Glutamate 47 is conserved in 1-aminocyclopropane-1-carboxylate (ACC) synthases and is positioned near the sulfonium pole of (S,S)-S-adenosyl-L-methionine (SAM) in the modeled pyridoxal phosphate quinonoid complex with SAM. E47Q 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 conversion 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 enzymes, 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-vinylglycine (L-VG) (deamination to form alpha-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 the enzyme [Feng, L., and Kirsch, J. F. (2000) Biochemistry 39, 2436-2444], but the partition ratio, k(cat)/k(inact), is unaffected by the Glu47 mutations. ACC synthase primarily catalyzes the beta,gamma-elimination of MTA 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 alpha,gamma-elimination of MTA. The partition ratios for (alpha,gamma/beta,gamma)-elimination on (R,S)-SAM are 0.4, < or =0.014, and < or =0.08 for the wild-type, E47Q, and E47D enzymes, respectively. The results of these experiments strongly support a role for Glu47 as an anchor for the sulfonium pole of (S,S)-SAM, and consequently a role as an active site determinant of reaction specificity.
McCarthy
D L
DL
Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, 229 Stanley Hall, University of California, Berkeley, 94720-3206, USA.
Capitani
G
G
Feng
L
L
Gruetter
M G
MG
Kirsch
J F
JF
D020134:Catalytic Domain / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry; D007700:Kinetics; D008190:Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010944:Plants / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000737:chemistry; D013379:Substrate Specificity; D014783:Viscosity
D011994:Recombinant Proteins; D018698:Glutamic Acid; D012436:S-Adenosylmethionine; D008190:Lyases; C021773:1-aminocyclopropanecarboxylate synthase