Shape Of Progress Curves Research Articles

Metal Ion Substrate Inhibition of Ferrochelatase

Ferrochelatase catalyzes the insertion of ferrous iron into protoporphyrin IX to form heme. Robust kinetic analyses of the reaction mechanism are complicated by the instability of ferrous iron in aqueous solution, particularly at alkaline pH values. At pH 7.00 the half-life for spontaneous oxidation of ferrous ion is approximately 2 min in the absence of metal complexing additives, which is sufficient for direct comparisons of alternative metal ion substrates with iron. These analyses reveal that purified recombinant ferrochelatase from both murine and yeast sources inserts not only ferrous iron but also divalent cobalt, zinc, nickel, and copper into protoporphyrin IX to form the corresponding metalloporphyrins but with considerable mechanistic variability. Ferrous iron is the preferred metal ion substrate in terms of apparent k(cat) and is also the only metal ion substrate not subject to severe substrate inhibition. Substrate inhibition occurs in the order Cu(2+) > Zn(2+) > Co(2+) > Ni(2+) and can be alleviated by the addition of metal complexing agents such as beta-mercaptoethanol or imidazole to the reaction buffer. These data indicate the presence of two catalytically significant metal ion binding sites that may coordinately regulate a selective processivity for the various potential metal ion substrates.

Exploration of the biocatalytic potential of a halohydrin dehalogenase using chromogenic substrates

Halohydrin dehalogenases are bacterial enzymes that catalyse the reversible formation of epoxides from vicinal halohydrins. A spectrophotometric assay for halohydrin dehalogenases based on the absorption difference between the halohydrin para-nitro-2-bromo-1-phenylethanol and the epoxide para-nitrostyrene oxide was developed. The enantioselectivity of ring-closure reactions catalysed by three different halohydrin dehalogenases could be estimated from the shape of progress curves. Evaluation of ring-opening reactions catalysed by halohydrin dehalogenase from Agrobacterium radiobacter AD1 established that, in addition to Cl − and Br −, nucleophiles such as N 3 −, CN − and NO 2 − are also accepted for the ring opening of para-nitrostyrene oxide. The ring-opening reactions with these nucleophiles resulted in highly enantioselective kinetic resolutions, which expands the scope of synthetically valuable conversions catalysed by a halohydrin dehalogenase.

Exploration systematique de la cinetique de rehydratation d'un sel renversable: II. Essai de generalisation des lois cinetiques

The rehydration kinetics of anhydrous compounds: calcium oxalate CaC 2O 4 and oxalic acid H 2C 2C 4, have been investigated according to temperature and water vapour pressure. Two essential criteria have been used in this paper: the shape of progress curves vs. time and the isothermal or isobaric laws of hydration rates. From the study of these criteria, two clearly distinct areas can be described within the stable region of the hydrated phase. The phenomena which regulate the hydration kinetics, following temperature and pressure conditions, in these two areas can be defined as: (a) Surface phenomena determine the kinetics in the region of low pressure and low temperature, and under the attraction of the hydrate/saturated solution equilibrium. (b) In an extended region, at high pressure and high temperature and under the attraction of the anhydrous/hydrate equilibrium, on the contrary, the volume phenomena become determining. (c) In the intermediate region, it seems that the hydration kinetics are regulated by the interface progression. The morphological study of solid grains accords with this analysis. A scheme which generalizes the behaviour of these solids during their hydration is proposed in conclusion.