Table 2.

Kinetic characterization of WT hASNase3

A. Kinetic parameters of WT hASNase3a
Asn			P-L-Asp-L-Phe
Km (mM)	kcat (S-1)	kcat/KM (mM-1s-1)	Km (mM)	kcat (S-1)	kcat/KM (mM-1s-1)
2.09 (± 0.16)	3.19 (± 0.07)	1.52 (± 0.15)	0.12 (± 0.02)	0.80 (± 0.06)	6.66 (± 1.61)

B. Enzyme concentrations ([E]) used to determine asparaginase activity
	% Cleavageb	Activityc	[E]d no correction	[E]e with correction
WT	100	Yes	75 nM Enzyme	75 nM Enzyme
T186V	45	Not detected	400 nM Enzyme	180 nM Enzyme
T219A	100	Not detected	310 nM Enzyme	310 nM Enzyme
T219V	50	Not detected	310 nM Enzyme	155 nM Enzyme

^aReactions were conducted at 37 °C, pH 7.5 and in triplicate.

^bDetermined by gel densitometry analysis

^cMeasured with [ASN] = 20 mM

^dTotal [E] in cuvette

^eApproximated cleaved [E] in cuvette