Uridine-cytidine kinase: Kinetic studies and reaction mechanism

Abstract

The initial velocity pattern has been determined for uridine-cytidine kinase purified from the murine mast cell neoplasm P815. With either uridine or cytidine as phosphate acceptor, and ATP as phosphate donor, the pattern observed was one of intersecting lines, ruling out a ping-pong reaction mechanism, and suggesting that the reaction probably proceeds by the sequential addition of both substrates to the enzyme to form a ternary complex, followed by the sequential release of the two products. This pattern was obtained whether the reaction was run in 0.01 m potassium phosphate buffer, pH 7.5, or in 0.1 m Tris-HCl, pH 7.2. When analyzed by the Sequen computer program, the data indicated an apparent K m of the enzyme for uridine of 1.5 × 10 −4 m, an apparent K m for cytidine of 4.5 × 10 −5 m, and a K m for ATP, with uridine or cytidine as phosphate acceptor, of 3.6 × 10 −3 m or 2.1 × 10 −3 m, respectively. The V was 1.83 μmol phosphorylated/min/mg enzyme protein for the uridine kinase reaction and 0.91 μmol for the cytidine kinase reaction.