Abstract
ATP sulfurylase from Penicillium chrysogenum is a noncooperative homooligomer containing three free sulfhydryl groups per subunit. Under nondenaturing conditions, one SH group per subunit was modified by 5,5'-dithiobis-(2-nitrobenzoate), or N-ethylmaleimide. Modification had only a small effect on kcat, but markedly increased the [S]0.5 values for the substrates, MgATP and SO4(2-). MgATP and adenosine-5'-phosphosulfate protected against modification. The SH-modified enzyme displayed sigmoidal velocity curves for both substrates with Hill coefficients (nH) of 2. Fluorosulfonate (FSO3-) and other dead-end inhibitors competitive with SO4(2-) activated the SH-modified enzyme at low SO4(2-) concentration. In order to determine whether the sigmoidicity resulted from true cooperative binding (as opposed to a kinetically based mechanism), the shapes of the binding curves were established from the degree of protection provided by a ligand against phenylglyoxal-dependent irreversible inactivation under noncatalytic conditions. Under standard conditions (0.05 M Na-N-(2-hydroxyethyl)piperazine-N'-3-propanesulfonic acid buffer, pH 8, 30 degrees C, and 3mM phenylglyoxal) the native enzyme was inactivated with a k of 2.67 +/- 0.25 X 10-3 s-1, whereas k for the SH-modified enzyme was 5.44 +/- 0.27 X 10-3 s-1. The increased sensitivity of the modified enzyme resulted from increased reactivity of ligand-protectable groups. Both the native and the SH-modified enzyme displayed hyperbolic plots of delta k (i.e. protection) versus [MgATP], or [FSO3-], or [S2O3(2-]) in the absence of coligand (nH = 0.98 +/- 0.06). The plots of delta k versus [ligand] for the native enzyme were also hyperbolic in the presence of a fixed concentration of coligand. However, in the presence of a fixed [FSO3-] or [S2O3(2-]), the delta k versus [MgATP] plot for the SH-modified enzyme was sigmoidal, as was the plot of delta k versus [FSO3-] or [S2O3(2-]) in the presence of a fixed [MgATP]. The nH values were 1.92 +/- 0.09. The results indicate that substrates (or analogs) bind hyperbolically to unoccupied SH-modified subunits, but in a subunit-cooperative fashion to form a ternary complex.
Highlights
ATP sulfurylase fromPenicillium chrysogenumis a noncooperative homooligomer containingthreefree sulfhydryl groups per subunit
Each subunit contains three free sulfhydryl groups, one of which can be readily modified under nondenaturing conditions
More recent studies disclosed that sulfhydryl modification markedly affected enzyme activity at subsaturating substrate concentrations[3]
Summary
Enzyme Purification and Assays-The purification of ATP sulfurylase and APS kinase, the assaymethods (3-lo), and the general procedures used to characterize the kinetics of inactivation in the presence and absence of protective ligands [11,12] have been described previously. Inactivation by Phenylglyoral-The native or NEM-modified enzyme (0.5 PM sites unless otherwise indicated) was preincubated for various periods in 0.05 M Na-EPPS buffer (pH 8.0, 30 “C)containing 3 mM phenylglyoxal, the protective ligands, and (unless otherwise indicated) 5 mM excess M$+. The variance in daily replicates using a freshly prepared phenylglyoxal solution each time was greater: kep, for the native enzyme averaged 2.7 X s-' with a maximum E deviation of k0.3 X s-'; kappfor the NEM-modified enzyme was. The Hill coefficient, nH, was obtained from the slope of the log [Ak/ (Akmex,a,,- Ak)] uersus log [L] plot
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