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Continuous coupled assay for 5-aminolevulinate synthase
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Shoolingin-Jordan, Peter M., LeLean, Jeremy E. and Lloyd, Adrian J. (1997) Continuous coupled assay for 5-aminolevulinate synthase. Methods in Enzymology, 281 . pp. 309-316. doi:10.1016/S0076-6879(97)81037-3 ISSN 00766879.
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Official URL: http://dx.doi.org/10.1016/S0076-6879(97)81037-3
Abstract
This chapter discusses the continuous coupled assay for 5-aminolevulinate synthase. A direct 5-aminolevulinate synthase enzyme assay has been developed on the basis of linking 5-aminolevulinate synthase to pyruvate dehydrogenase. Initial investigations demonstrated that pyruvate dehydrogenase was unaffected by components of the 5-aminolevulinate synthase assay system and vice versa. When 5-aminolevulinate synthase activity was determined using this coupled assay, a linear increase in absorbance was obtained. The rate was dependent on the presence of both succinyl-CoA and glycine and was linear with respect to both time and protein concentration. A Lineweaver–Burk plot of dependence of activity of the Rhodobacter spheroides enzyme on succinyl-CoA gave values for Km and Vmax of 6μM and 138μmol/ mg protein/hour, respectively. The Km for glycine was 5 mM. These values agree well with those in the literature. Data obtained using the coupled assay showed excellent agreement with the data obtained using the discontinuous assay.
| Item Type: | Journal Article | ||||||
|---|---|---|---|---|---|---|---|
| Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) | ||||||
| Journal or Publication Title: | Methods in Enzymology | ||||||
| Publisher: | Elsevier Science BV | ||||||
| ISSN: | 00766879 | ||||||
| Book Title: | Vitamins and Coenzymes Part K | ||||||
| Official Date: | 4 June 1997 | ||||||
| Dates: |
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| Volume: | 281 | ||||||
| Page Range: | pp. 309-316 | ||||||
| DOI: | 10.1016/S0076-6879(97)81037-3 | ||||||
| Status: | Not Peer Reviewed | ||||||
| Publication Status: | Published | ||||||
| Access rights to Published version: | Restricted or Subscription Access |
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