Adenosine 5′-Phosphosulfate (APS) Kinase: Diagnosing the Mechanism of Substrate Inhibition

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 thevversus [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]optdecreases asymptotically to a limit as the fixed [MgATP] is increased, while in the competitive system, [APS]optincreases 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 ofv/voptversus [APS] clearly display these distinguishing characteristics. The method confirmed thatPenicillium chrysogenumAPS kinase exhibits uncompetitive inhibition by APS.