Expression of apple 1-aminocyclopropane-1-carboxylate synthase in Escherichia coli: kinetic characterization of wild-type and active-site mutant forms.

White, Malcolm F.; Vásquez, John R.; Yang, Shang Fa; Kirsch, Jack F.

doi:10.1073/pnas.91.26.12428

Cited by 80 publications

(80 citation statements)

References 32 publications

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“…Kinetic Characterization of ACC Synthase Inactivation by AVG-ACC synthase activity in the presence of AVG was monitored by a continuous coupled assay (14). Reactions were initiated by the addition of ACC synthase to mixtures of AVG, SAM, and coupling enzyme preequilibrated in buffer at 25°C.…”

Section: Methodsmentioning

confidence: 99%

Apple 1-Aminocyclopropane-1-carboxylate Synthase in Complex with the Inhibitor l-Aminoethoxyvinylglycine

Capitani¹,

McCarthy²,

Gut³

et al. 2002

Journal of Biological Chemistry

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The 1.6-Å crystal structure of the covalent ketimine complex of apple 1-aminocyclopropane-1-carboxylate (ACC) synthase with the potent inhibitor L-aminoethoxyvinylglycine (AVG) is described. ACC synthase catalyzes the committed step in the biosynthesis of ethylene, a plant hormone that is responsible for the initiation of fruit ripening and for regulating many other developmental processes. AVG is widely used in plant physiology studies to inhibit the activity of ACC synthase. The structural assignment is supported by the fact that the complex absorbs maximally at 341 nm. These results are not in accord with the recently reported crystal structure of the tomato ACC synthase AVG complex, which claims that the inhibitor only associates noncovalently. The rate constant for the association of AVG with apple ACC synthase was determined by stopped-flow spectrophotometry (2.1 ؋ 10 5 M ؊1 s ؊1 ) and by the rate of loss of enzyme activity (1

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Section: Methodsmentioning

confidence: 99%

Apple 1-Aminocyclopropane-1-carboxylate Synthase in Complex with the Inhibitor l-Aminoethoxyvinylglycine

Capitani¹,

McCarthy²,

Gut³

et al. 2002

Journal of Biological Chemistry

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“…In aspartate aminotransferase and 1-aminocyclopropane-1-carboxylate synthase, the phenolic oxygen interacts with a tyrosine residue (14,15). The deletion of the hydrogen bond through the replacement of the active site tyrosine with phenylalanine reveals a different function in the kinetic properties of each enzyme.…”

Section: Introductionmentioning

confidence: 99%

Histidine 282 in 5-Aminolevulinate Synthase Affects Substrate Binding and Catalysis

et al. 2007

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Abstract5-Aminolevulinate synthase (ALAS), the first enzyme of the heme biosynthetic pathway in mammalian cells, is member of the α-oxoamine synthase family of pyridoxal 5′-phosphate (PLP)-dependent enzymes. In all structures of the enzymes of the α-oxoamine synthase family, a conserved histidine hydrogen bonds with the phenolic oxygen of the PLP cofactor and may be significant for substrate-binding, PLP-positioning, and maintaining the pK a of the imine nitrogen. In ALAS, replacing the equivalent histidine, H282, with alanine reduces the catalytic efficiency for glycine 450-fold and decreases the slow phase rate for glycine binding by 85%. The distribution of the absorbing 420 and 330 nm species was altered with an increased A420/A330 ratio from 0.45 to 1.05. This shift in species distribution was mirrored in the cofactor fluorescence and 300 to 500 nm circular dichroic spectra and likely reflects variation in the tautomer distribution of the holoenzyme. The 300 to 500 nm circular dichroic spectra of ALAS and H282A diverged in the presence of either glycine or aminolevulinate indicating that the reorientation of the PLP cofactor upon external aldimine formation is impeded in H282A. Alterations were also observed in the value and spectroscopic and kinetic properties, while the increased 9-fold. Altogether, the results imply that H282 coordinates the movement of the pyridine ring with the reorganization of the active-site hydrogen bond network and acts as a hydrogen bond donor to the phenolic oxygen to maintain the protonated Schiff base and enhance the electron sink function of the PLP cofactor.

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“…The AdoMet used in the enzyme assay which contained 59% of (S,S) diastereomer form [9] was purchased from Boerhinger-Mannheim Biochem (Germany). ACC synthase activity was assayed by incubating the crude (5-15 pg) or the highly purified (0.3 pg) ACC synthase extract with AdoMet at 30°C for 30 min in a reaction buffer containing 25 mM HEPES at pH 8.5, 10 pM PLP and 1 mM DTT.…”

Section: N-terminal Deletion Nestmentioning

confidence: 99%

“…SSD1 0014-5793(95)01464-0 On optimum alignment of ACC synthase amino acid sequences, it was found that there were seven highly conserved regions [6] and 16 invariant amino acid residues [9] in this enzyme. The overall amino acid sequences are 72-81% similar and 53-67% identical with the exception of the amino-and, particularly, the carboxyl-termini of ACC synthase which were found to be highly variable in both length and primary sequence [10].…”

Section: Introductionmentioning

confidence: 99%

“…Expression of the full-length polypeptides of apple, zucchini, tomato and squash ACC synthases in a heterologous E. coli system has produced a functional enzyme. The molecular weights of these full-length polypeptides, as determined by SDS-PAGE analysis, are 52, 53, 54 and 60 kDa, respectively [4,9,10,11]. They are 4-10 kDa larger than the same enzyme directly isolated from plant tissue [2,11,12,13,14].…”

Section: Introductionmentioning

confidence: 99%

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Effects of N‐terminal deletions on 1‐aminocyclopropane‐1‐carboxylate synthase activity

Huxtable

Yang

et al. 1996

FEBS Letters

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A series of nested N-terminal deletions were made on the full-length (wt) and C-terminal deleted (Cdel) 1-aminocyclopropane-l-carboxylate synthase cDNAs. These wt and mutant ACC synthases were over-expressed in a heterologous E. coil expression system. It was found that removal of an amino acid region (residues 2-12) from the non-conserved N-termini of wt and Cdel ACC synthases led to a slight increase in both in vivo ACC production and in vitro ACC synthase activity. Further deletion of 11 amino acids through GIn-23 from the N-termini of both wt and Cdel ACC synthases resulted in a substantial reduction in both in vivo ACC production and in vitro enzyme activity. Deletion of an amino acid region, residues 3 through 27, from the N-terminus of ACC synthase abolished enzyme activity completely. Kinetic analysis of a highly purified double-deletion mutant (NCdel-1) of ACC synthase demonstrated that the Km of this mutant is 42 ~M, which is much smaller than that of the corresponding Cdel (280 /tM) and closer to that of wt (22 tiM) reported previously, suggesting a clear effect of the non-conserved N-terminal region on its ACC synthase function.

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Expression of apple 1-aminocyclopropane-1-carboxylate synthase in Escherichia coli: kinetic characterization of wild-type and active-site mutant forms.

Cited by 80 publications

References 32 publications

Apple 1-Aminocyclopropane-1-carboxylate Synthase in Complex with the Inhibitor l-Aminoethoxyvinylglycine

Apple 1-Aminocyclopropane-1-carboxylate Synthase in Complex with the Inhibitor l-Aminoethoxyvinylglycine

Histidine 282 in 5-Aminolevulinate Synthase Affects Substrate Binding and Catalysis

Effects of N‐terminal deletions on 1‐aminocyclopropane‐1‐carboxylate synthase activity

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