Purification of two forms of enoyl-CoA hydratase from Mycobacterium smegmatis

Two forms of enoyl-CoA hydratase (hydratases I and II), which are different from each other in substrate specificity, were found in a crude extract of Mycobacterium smegmatis. Hydratase I was more active with crotonyl-CoA as a substrate than with decenoyl-CoA, whereas the reverse was the case for hydratase II. Hydratase I was purified 688-fold to homogeneity with a yield of 14.5% from the crude extract. Its molecular weight was estimated to be 16,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 30,000 by gel filtration, suggesting that the enzyme is dimeric. Hydratase II was also partially purified. The Vmax of hydratase I decreased progressively with increase in the carbon-chain length of the substrate from 2,488 units/mg for crotonyl-CoA to 154 units/mg for hexadecenoyl-CoA, whereas the Km values for crotonyl-CoA (82 microM), decenoyl-CoA (91 microM), and hexadecenoyl-CoA (105 microM) were similar. Both hydratases were inhibited by acetoacetyl-CoA and pCMS, but not by N-ethylmaleimide or monoiodoacetate.