Characterization of two 3-ketothiolases possessing differing substrate specificities in the polyhydroxyalkanoate synthesizing organism Alcaligenes eutrophus |
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Authors: | GW Haywood AJ Anderson L Chu EA Dawes |
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Institution: | Department of Biochemistry, School of Life Sciences, University of Hull, Hull, U.K. |
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Abstract: | Abstract Two constitutive acetyl-CoA acetyltransferases (3-ketothiolases A and B) were purified from Alcaligenes eutrophus . Enzyme A was active with only acetoacetyl-CoA and 3-ketopentanoyl-CoA, whereas enzyme B was active with all the 3-ketoacyl-CoAs (C4?C10) tested. Enzyme A appeared to be a tetramer ( M r 70 000) with identical subunits ( M r 44 000) and enzyme B had a similar M r of 168 000 (containing M r 46 000 subunits). Enzymes A and B had isoelectric points of 5.0 and 6.4, respectively. The stoichiometry of the reactions catalysed by each enzyme was confirmed. K m values of 44 μM and 394 μM for acetoacetyl-CoA, and 16 μM and 93 μM for CoA, were determined with enzymes A and B, respectively. Enzymes A and B gave K m values of 1.1 mM and 230 μM, respectively, for acetyl-CoA. The condensation reaction was potently inhibited by CoA in both cases. |
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Keywords: | Alcaligenes eutrophus 3-Ketothiolase acetyl-CoA acetyltransferase |
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