|
type |
Journal Article |
authors |
Dowhan W Jr, Snell EE. |
title |
D-serine dehydratase from Escherichia coli. II. Analytical studies and subunit structure |
journal |
J Biol Chem |
Activity |
4.3.1.18 |
sel |
selected |
ui |
4917239 |
year |
(1970) |
volume |
245 |
number |
18 |
pages |
4618-28 |
| |
abstract |
An improved procedure which permits large scale preparation of crystalline D-serine dehydratase (EC 4.2.1.14) from Escherichia coli is described. The homogeneous holoenzyme has an extinction coefficient (E 1%) of 10.5 in potassium phosphate, pH 6.5 to 7.8, a specific activity of 300, and contains a single peptide chain with a molecular weight near 45,500 as indicated by sedimentation equilibrium measurements of either native or reduced and carboxyxnethylated enzyme, acrylamide gel electrophoresis in sodium dodecyl sulfate, quantitative determination of the N-terminal amino acid (methionine), and the results of tryptic peptide mapping. One combining site for pyridoxal-P is present per molecule as determined by direct analysis of the holoenzyme for pyridoxal-P, by spectrophotometric titration of the apoenzyme with pyridoxal-P, and by isolation of e-N-pyridoxyllysine from hydrolysates of the sodium borohydride-reduced holoenzyme. The enzyme contains 5 half-cystine residues and no disulfide linkages. Only one of the five sulfhydryl groups is titrated by 5,5’-dithio-bis(Z-nitrobenzoic acid) (DTNB) in the holoenzyme; in the apoenzyme three -SH groups are titrated by either p-chloromercuribenzoate or DTNB. These three groups react with DTNB at markedly different rates (0.54, 0.081, and 0.0033 min-l) under the conditions tested. Individually, none of the three -SH groups appears necessary for activity; however, reaction of two or more with DTNB inactivates the apoenzyme completely by preventing its reassociation with pyridoxal-P. |
last changed |
2009/06/30 14:57 |
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