Adenosine 5'-phosphosulfate (APS) kinase: diagnosing the mechanism of substrate inhibition |
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Authors: | IJ MacRae IH Segel |
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Affiliation: | Section of Molecular and Cellular Biology, University of California, One Shields Avenue, Davis, California, 95616, USA. |
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Abstract: | Adenosine 5'-phosphosulfate (APS) kinase is subject to strong substrate inhibition by APS. The inhibition has been variously reported to be uncompetitive with respect to MgATP (resulting from the formation of a dead-end E. APS. MgADP complex) or competitive with MgATP (resulting from the formation of a dead-end E. APS complex). It is shown that these two types of substrate inhibition can be differentiated for ordered kinetic mechanisms by simple inspection of the v versus [APS] plots at different fixed concentrations of MgATP. Linear diagnostic plots are unnecessary. One diagnostic feature is the changing position of [APS]opt, the concentration of APS that yields the peak velocity. In the uncompetitive system, [APS]opt decreases asymptotically to a limit as the fixed [MgATP] is increased, while in the competitive system, [APS]opt increases continuously as the fixed [MgATP] is increased. A second (and more easily discerned) diagnostic feature is that, at any given inhibitory level of APS, enzyme activity relative to the velocity at [APS]opt (v/vopt) decreases as the fixed [MgATP] is increased in the uncompetitive system, while in the competitive system the relative activity increases as the fixed [MgATP] is increased. Normalized plots of v/vopt versus [APS] clearly display these distinguishing characteristics. The method confirmed that Penicillium chrysogenum APS kinase exhibits uncompetitive inhibition by APS. |
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