Abstract
1. L-Serine dehydratase (EC 4.2.1.13) was purified 970-fold from glycine-grown Arthrobacter globiformis to a final specific activity of 660micronmol of pyruvate formed/min per mg of protein. 2. The enzyme is specific for L-serine; D-serine, L-threonine and L-cysteine are not attacked. 3. The time-course of pyruvate formation by the purified enzyme, in common with enzyme in crude extracts and throughout the purification, is non-linear. The reaction rate increases progressively for several minutes before becoming constant. The enzyme is activated by preincubation with L-serine and a linear time-course is then obtained. 4. The substrate-saturation curve for L-serine is sigmoid. The value of [S]0.5 varies with protein concentration, from 6.5mM at 23microng/ml to 20mM at 0.23microng/ml. The Hill coefficient remains constant at 2.9.5 The enzyme shows a non-specific requirement for a univalent or bivalent cation. Half-maximal activity is produced by 1.0mM-MgCl2 or by 22.5mM-KCl. 6. L-Cysteine and D-serine act as competitive inhibitors of L-serine dehydratase, with Ki values of 1.2 and 4.9mM respectively. L-Cysteine, at higher concentrations, also causes a slowly developing irreversible inhibition of the enzyme. 7. Inhibition by HgCl2 (5micronM)can be partially reversed in its initial phase by 1mM-L-cysteine, but after 10 min it becomes irreversible. 8. In contrast with the situation in all cell-free preparations, toluene-treated cells of A. globiformis form pyruvate from L-serine at a constant rate from the initiation of the reaction, show a hyperbolic substrate-saturation curve with an apparent Km of 7mM and do not require a cation for activity.
Highlights
The present paper describes the purification of a specific L-serine dehydratase (EC 4.2.1.13) from the aerobic soil bacterium, Arthrobacter globiformis, and compares the properties of the purified enzyme with those observed with toluene-treated whole cells
When cells of A. globiformis, grown on glycine as source of carbon and nitrogen, were toluene-treated and incubated in a mixture containing L-serine and buffer, pyruvate was formed at a rate that was constant for at least 30min and was directly proportional to the quantity of cells added to the incubation mixture
In a number ofrespects the L-serine dehydratase of A. globiformis shows characteristics that are found in enzymes of this type derived from other sources
Summary
L-Serine (chromatographically homogeneous) was obtained from BDH Chemicals Ltd., Poole, Dorset, U.K.; D-serine from Koch-Light Laboratories, F. The mixtures were left for 5 min at room temperature, 2ml of 2 M-NaOH was added to each, and after a further 10min the A445 was measured This method was used for experiments with toluene-treated cells (in which case MgCl2 was omitted from the assay mixture), with crude preparations containing high NADH oxidase activity, in experiments where large numbers of enzyme samples were to be assayed, e.g. column fractions, and when substances that would inhibit lactate dehydrogenase, e.g. mercurial compounds, were present in the assay mixture. MgCl2 and D-serine were added to the concentrated enzyme solution from step 5 to give final concentrations of 10 mM and 150 mmrespectively, and the mixture was incubated at 15°C for 15min. Fractions (5ml) having a specific activity greater than 400,umol/min per mg of protein were pooled, concentrated by ultrafiltration, and stored at -70°C
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