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Haloalkane dehalogenase converts halogenated alkanes to their corresponding alcohols. The active site is buried inside the protein and lined with hydrophobic residues. The reaction proceeds via a covalent substrate-enzyme complex. This paper describes a steady-state and pre-steady-state kinetic analysis of the conversion of a number of substrates of the dehalogenase. The kinetic mechanism for the "natural" substrate 1,2-dichloroethane and for the brominated analog and nematocide 1,2-dibromoethane are given. In general, brominated substrates had a lower Km, but a similar kcat than the chlorinated analogs. The rate of C-Br bond cleavage was higher than the rate of C-Cl bond cleavage, which is in agreement with the leaving group abilities of these halogens. The lower Km for brominated compounds therefore originates both from the higher rate of C-Br bond cleavage and from a lower Ks for bromo-compounds. However, the rate-determining step in the conversion (kcat) of 1, 2-dibromoethane and 1,2-dichloroethane was found to be release of the charged halide ion out of the active site cavity, explaining the different Km but similar kcat values for these compounds. The study provides a basis for the analysis of rate-determining steps in the hydrolysis of various environmentally important substrates.

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The growth of Pseudomonas oleovorans on n-octane was characterized by the formation of intracellular structures. These inclusions were isolated and characterized. Morphologically, they resembled the poly-p-hydroxybutyrate granules found in Bacillus cereus, as shown by freeze-fracture electron microscopy. The elemental analysis of isolated granules showed, however, that they do not contain poly-4-hydroxybutyric acid. Instead, the analysis was consistent with a C8 polyester, which interpretation was supported by the fatty acid analysis of hydrolyzed granules. From the evidence presented here, we conclude that P. oleovorans forms poly-p-hydroxyoctanoate granules when grown on n-octane. MATERIALS AND METHODS Chemicals. n-Octane (>99% pure) was purchased from J. T. Baker Chemical Co., Phillipsburg, N.J. A standard mixture (4-5436) of fatty acid methyl esters was purchased from Supelco, Inc., Bellefonte, Pa.; stearic methyl ester was purchased from Merck-Schuchardt A. G.; and egg white lysozyme was purchased from Boehringer Mannheim GmbH and Soehne GmbH. Rnase I (EC 3.1.4.22) was purchased from Miles-Seravac Ltd., and DNase I (EC 3.1.4.5) was purchased from Sigma Chemical Co.