Figure 5.

Phosphorylation of Lck catalyzed by semisynthetic Csk proteins. (A) Time course of Csk^PEP and Csk^pPEP-catalyzed phosphorylation of Lck. •, Csk^pPEP; ○, Csk^PEP. Relative product formation is corrected for the amount of Csk^PEP and Csk^pPEP utilized. [Lck] = 1 μM. (B) Kinase activity vs. Csk^PEP and Csk^pPEP concentration with the substrate Lck. •, Csk^pPEP; ○, Csk^PEP. [Lck] = 1 μM. Reaction time = 2 min. (C) Rate of phosphorylation of Lck catalyzed by Csk^PEP and Csk^pPEP vs. Lck concentration. •, Csk^pPEP; ○, Csk^PEP. The data for Csk^pPEP could not be strictly fit to standard Michaelis-Menten kinetics because of apparent substrate inhibition. For Csk^pPEP the K_m (apparent) (Lck) estimated as the substrate concentration necessary for 50% maximal velocity is 0.4 ± 0.1 μM. The data for Csk^PEP was fit to the Michaelis-Menten equation and gave K_m (apparent) (Lck) = 2 ± 0.5 μM. The Lck K_m with wild-type Csk enzyme under these conditions is 3–5 μM (25). All assays were performed as described (25) on a 15 μl scale at 30°C by using catalytically impaired Lck (N-terminal 63 aa deleted, containing a K273R mutation) at fixed and near-saturating ATP concentration [10 μM, ≈3 × K_m (apparent)] and optimal MnCl₂ concentration (5 mM) under initial conditions (<10% turnover of the limiting substrate). All assays were carried out at least two times and duplicate points generally agreed within 10%. Kinase activity was shown to be identical for the phosphotyrosine-affinity purified and unpurified semisynthetic proteins by using Lck as substrate. Kinase specificity for the 505-tyrosine of Lck was confirmed by demonstrating that 505-phosphorylated Lck was not detectably phosphorylated by either semisynthetic protein using conditions described (25).