D-Amino Acid Oxidase

Cyclopropane fatty acid (CFA) synthase of Escherichia coli catalyzes a modification of the acyl chains of phospholipid bilayers. We report (i) identification of the CFA synthase protein, (ii) overproduction (> 600-fold) and purification to essential homogeneity of the enzyme, and (iii) the amino acid sequence of CFA synthase as deduced from the nucleotide sequence of the cfa gene. CFA synthase was overproduced by use of the T7 promoter/RNA polymerase system under closely defined conditions. The enzyme was readily purified by a two-step procedure requiring only ammonium sulfate fractionation and binding to phospholipid vesicles followed by flotation in sucrose density gradients. The deduced amino acid sequence predicts a protein of 43,913 Da (382 residues) that lacks long hydrophobic segments. The CFA synthase sequence has no significant similarity to known proteins except for sequences found in other enzymes that utilize S-adenosyl-L-methionine. We also report inhibitor studies of the enzyme active site.

Section: Resultsmentioning

confidence: 99%

Cyclopropane fatty acid synthase of Escherichia coli: Deduced amino acid sequence, purification, and studies of the enzyme active site

Wang¹,

Grogan²,

Cronan³

1992

“…The different kinetic behavior of DTNB and NEM with respect to inactivation of glycerol kinase may reflect different affinities for a binding site near the modified group. Saturation kinetics have been observed in the modification of bovine serum albumin with fluorodinitrobenzene (Green, 1963) and in the modification of o-amino acid oxidase with TV-alkylmaleimides (Fonda & Anderson, 1969) or 1,2-cyclohexanedione (Ferti et al, 1981). In each of these cases, the observed saturation kinetics has been interpreted in terms of a two-step mechanism for the modification, where the first step is binding of the reagent to a nonpolar region of the protein in the vicinity of the modified amino acid.…”

Section: Discussionmentioning

confidence: 99%

“…The apparent absence of binding of NEM may reflect differences in polarity and/or size. If binding of DTNB is due to the aromatic ring, then saturation kinetics may be observed with other N-substituted maleimides, as is the case with damino acid oxidase (Fonda & Anderson, 1969). On the other hand, the binding of DTNB may involve electrostatic interactions with the ionized carboxyl groups on the reagent.…”

Section: Discussionmentioning

confidence: 99%

Inactivation of Escherichia coli glycerol kinase by 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide: evidence for nucleotide regulatory binding sites

Pettigrew¹

1986

Glycerol kinase (EC 2.7.1.30, ATP:glycerol 3-phosphotransferase) from Escherichia coli is inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and by N-ethylmaleimide (NEM) in 0.1 M triethanolamine at pH 7 and 25 degrees C. The inactivation by DTNB is reversed by dithiothreitol. In the cases of both reagents, the kinetics of activity loss are pseudo first order. The dependencies of the rate constants on reagent concentration show that while the inactivation by NEM obeys second-order kinetics (k2app = 0.3 M-1 s-1), DTNB binds to the enzyme prior to the inactivation reaction; i.e., the pseudo-first-order rate constant shows a hyperbolic dependence on DTNB concentration. Complete inactivation by each reagent apparently involves the modification of two sulfhydryl groups per enzyme subunit. However, analysis of the kinetics of DTNB modification, as measured by the release of 2-nitro-5-thiobenzoate, shows that the inactivation is due to the modification of one sulfhydryl group per subunit, while two other groups are modified 6 and 15 times more slowly. The enzyme is protected from inactivation by the ligands glycerol, propane-1,2-diol, ATP, ADP, AMP, and cAMP but not by Mg2+, fructose 1,6-bisphosphate, or propane-1,3-diol. The protection afforded by ATP or AMP is not dependent on Mg2+. The kinetics of DTNB modification are different in the presence of glycerol or ATP, despite the observation that the degree of protection afforded by both of these ligands is the same.(ABSTRACT TRUNCATED AT 250 WORDS)

“…Amino acid analysis was performed as described previously (Swenson et al, 1982). The color constant used for Ssuccinylcysteine (emerging just ahead of aspartic acid) was 1.08 times that for aspartic acid (Fonda & Anderson, 1969). Three microliters of ethanethiol was contained in the hydrolysis of NEM-modified protein.…”

Section: Spectral Characterization Of Nem-modified Thioredoxinmentioning

confidence: 99%

Reaction of both active site thiols of reduced thioredoxin reductase with N-ethylmaleimide

O’Donnell¹,

Williams²

1985

Thioredoxin reductase from Escherichia coli, only in its reduced state, reacts rapidly with 2 mol of N-ethylmaleimide, which specifically alkylates both active site cysteine residues. This dual modification supports previous studies indicating that a base lowers the pK of both active site cysteine residues. The dual modification also indicates that the region around the active site dithiol is more open than is the case with the related enzymes lipoamide dehydrogenase and glutathione reductase, both of which can be alkylated only on one nascent thiol. Enhanced nucleophilicity of the active site thiols is consistent with the proposed chemical mechanism of thioredoxin reductase. The sequence of the amino-terminal 16 residues is presented.