Role of gamma-carboxyglutamic acid. An unusual protein transition required for the calcium-dependent binding of prothrombin to phospholipid.

A first order calcium-dependent transition can be monitored by a decrease in the intrinsic fluorescence of the isolated "pro" (Fragment1) region of prothrombin. The maximum fluorescence change is -40% for Fragment 1, and only about -6% for whole prothrombin. The most remarkable features of this transition are its rate and activation energy. The half-life for the transition at 0 degrees is about 100 min, and the temperature dependence shows an activation energy of 21 kcal/mol. The rate constant for the forward reaction is zero order in calcium and is not affected by the presence of phospholipid membranes. The equilibrium for the transition, however, is affected by phospholipid. At 30 degrees, Caeq (the calcium concentration where half of the protein has undergone the transition) is 0.4 mM and the Hill coefficient is 2.6. Under the same conditions but in the presence of phospholipid Caeq is 0.24 mM and the Hill coefficient is about 4.5. The transition is triggered by binding 3 or 4 calcium ions. The rate of Fragment 1 binding to phospholipid vesicles was tested using gel filtration techniques at 0 degrees. The rate constants, activation energy, and Caeq values for this process were shown to correspond to the properties of the fluorescence change. The rate constants, activation energy, and Hill coefficients for binding of whole prothrombin to phospholipid correspond to the same parameters for Fragment 1 but the Caeq values are lower. At 0 degrees, the Caeq is 0.19 mM for the prothrombin transition and 0.1 mM for the transition in the presence of phospholipid. These results demonstrate that Fragment 1 and prothrombin undergo a transition when exposed to calcium ions which necessarily precedes protein-phospholipid interactions. In addition to its role in determining the correct protein structure, calcium plays a second role in prothrombin-phos-pholipid interaction which is in the actual formation of the protein-phospholipid bond. The Caeq for binding protein (after its transition) to phospholipid is about 0.06 mM.