TABLE 4.

αSNAP₃ activation of NSF-mediated ATP hydrolysis

NSF ATP hydrolysis rates were measured as a function of increasing ATP concentration (0–600 μm) and fit to the Michaelis-Menten equation using nonlinear regression with [NSF hexamer] = 4–8 nm, [αSNAP] = 4.5 μm, [αSNAP₃] = 1.5 μm, and [αSNAP₃·SC] = 2 μm. At these concentrations, which were limited by protein solubility, αSNAP and αSNAP₃ in the absence of SC do not fully saturate NSF. Specifically, there is predicted to be 50% free NSF and 50% NSF·αSNAP in the experiment for NSF·αSNAP and 19% free NSF and 81% NSF·αSNAP₃ in the experiment for NSF·αSNAP₃ (calculated using K_m for αSNAP and αSNAP₃ stimulation of ATPase activity; Fig. 2, D and E, respectively), and the k_cat values have been corrected for these fractions bound using the ATP hydrolysis rate for NSF alone for the αSNAP-free fraction in this table.

	NSF	NSF·αSNAP	NSF·αSNAP3	NSF·αSNAP3·SC
kcatATP (min−1)	5.7 ± 0.21	10.3 ± 0.25; 14.9 (corrected)	40.0 ± 3.6; 48.1 (corrected)	77.2 ± 3.9
KmATP (μm)	20 ± 3.6	46.6 ± 4.6 (uncorrected)	56.9 ± 20 (uncorrected)	52.9 ± 9.0
kcatATP/KmATP (μm−1 min−1)	0.29 ± 0.05	0.22 ± 0.02	0.70 ± 0.26	1.46 ± 0.26