Nucleoside phosphotransferase from carrot. Kinetic studies and exploration of active sites.

In the light of the kinetic studies reported in this paper, the nucleoside phosphotransferase of carrot is considered as an enzyme possessing two hydrolytic sites, binding the phosphate donor, and one transfer site, engaging the nucleoside acceptor. The purification of the protein used in these studies, with phenylphosphate as the donor and uridine as the acceptor, is described. In addition to phenylphosphate, D-ribose 5-phosphate was also used as the substrate in the study of the hydrolytic function of the enzyme. Michaelis constants for the transfer reaction were determined with phenylphosphate as the donor (K, = 3.5 mM) and uridine as the acceptor (K, = 3.5 mrvr). As concerns the hydrolase function, either substrate exhibited two K, values: approximately 0.7 and 3 mM for phenylphosphate, and approximately 2 and 60 mM for ribose phosphate. Uridine acted as a noncompetitive inhibitor of the hydrolase function, with Ki = 4.3 mM. Ribose phosphate produced a “mixed type” inhibition. The evidence pointing to one protein carrying both transfer and hydrolytic functions may be summarized as follows: (a) the constant specific activity of a single, homogeneous protein peak in the chromatographic eluates; (b) similar constants for uridine as acceptor of phosphate transfer and as inhibitor of hydrolysis; (c) the demonstration, in the two separate enzymic functions, of two ionizing groups in the free enzyme having identical pK values (7.0, 7.6); (d) the similarity of the pKEs values recorded for the enzyme-substrate complexes in both enzymic functions. With regard to the suggestion that the enzyme possesses two hydrolytic centers, it rests, among other observations discussed in this communication, on two points: (a) the nonlinearity of the Lineweaver-Burk plots depicting the hydrolysis of phenylphosphate and ribose 5-phosphate; (b) the existence, in the free enzyme, of two ionizing groups (pK 7.0, 7.6), both binding phenylphosphate in the course of its hydrolysis.