ATP sulfurylase from Penicillium chrysogenum. Molecular basis of the sigmoidal velocity curves induced by sulfhydryl group modification.
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.
Renosto
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Department of Biochemistry and Biophysics, University of California, Davis 95616.
Martin
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Segel
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D000255:Adenosine Triphosphate; D004228:Dithionitrobenzoic Acid; D005459:Fluorides; D007700:Kinetics; D009713:Nucleotidyltransferases / Q000378:metabolism*; D010407:Penicillium / Q000201:enzymology*; D010408:Penicillium chrysogenum / Q000201:enzymology*; D012966:Sodium Cyanide; D013329:Structure-Activity Relationship; D013430:Sulfate Adenylyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013438:Sulfhydryl Compounds; D013464:Sulfuric Acids
D013438:Sulfhydryl Compounds; D013464:Sulfuric Acids; D000255:Adenosine Triphosphate; D004228:Dithionitrobenzoic Acid; D009713:Nucleotidyltransferases; D013430:Sulfate Adenylyltransferase; D012966:Sodium Cyanide; C046106:fluorosulfonic acid; D005459:Fluorides