Figure 5.

Phosphorylation of Xic1 by cyclin E is second order with respect to concentration. (A) Schematic models describing mechanisms by which cyclin E/Cdk2 might phosphorylate Xic1. Mechanism 1 is an intracomplex interaction (first order), whereas mechanisms 2 and 3 are intercomplex interactions (second order). (B) Cyclin E/Cdk2 and MBP-Xic1 were mixed in equimolar amounts. After 30 min at room temperature, the reaction was diluted to the indicated concentrations, and kinase reactions were initiated, processed, and analyzed as described in Materials and Methods. Values for Xic1 phosphorylation were normalized by concentration and fit to a linear curve (R² = 0.99). (C) Varying concentrations of MBP-Xic1 were incubated with a fixed concentration of cyclin E/Cdk2 (400 nM). After 30 min the Xic1 phosphorylation reactions were initiated, processed, and analyzed as in A except that the counts were plotted versus the concentration squared through and fit to a linear curve (R² = 0.97).