Table 1.

GST–XD4 and GST–XD4(V323A, I338A) catalyzed RR-Src peptide phosphorylation kinetic constants

Nucleotide	GST-XD4			GST-XD4 (V323A, I338A)
	kcat, min−1	Km, μM	kcat/Km, min−1·M−1	kcat, min−1	Km, μM	kcat/Km, min−1·M−1
ATP	2 ± 0.5	12 ± 3	1.6 × 105	0.8 ± 0.2	150  ±  20	5.3  ×  103
N6-(cyclopentyl) ATP		>2000 (Kl)		(5 ± 2) × 10−2	15  ±  3	3.3  ×  103

ATP and N⁶-(cyclopentyl) ATP-dependent RR-Src phosphorylation (1 mM) by GST–XD4(V323A, I338A) and GST–XD4 was measured at low substrate conversion (<5%) in triplicate. Kinetic constants were determined by analysis of Lineweaver–Burk plots of the rate data (41). Reactions were performed in the same manner as in Fig. 4B except for substitution of [γ-³²P]ATP with [γ-³²P]N⁶-(cyclopentyl) ATP (5000 cpm/pmol) as indicated.