Molecular and biochemical characterization of key enzymes in the cysteine and serine metabolic pathways of Acanthamoeba castellanii

Table 2 Kinetic parameters of AcCS, AcGDH and AcSPAT

Recombinant proteins	Substrate/cofactor	Km (mM)	Specific activity (mmol/min per mg of protein)	kcat (^-s)
AcCS	O-acetylserine	0.39 ± 0.02	63.92 ± 6.33
AcCS	Na₂S	1.44 ± 0.08	0.28 ± 0.01	2.36 × 10⁴ ± 978
AcGDH	Hydroxypyruvate	17.93 ± 1.71	26.50 ± 0.85
	NADPH	1.01 ± 0.06	2.05 ± 0.04	9.5 × 10³ ± 297
	Glycerate	213.80 ± 19.77	20.48 ± 0.65
	NADP+	19.90 ± 0.74	20.85 ± 0.17	1.38 × 10⁴ ± 273
AcSPAT	Pyruvate	5.26 ± 0.40	6.98 ± 0.18
AcSPAT	Serine	0.72 ± 0.03	13.17 ± 0.19	6.66 × 10³ ± 122

The enzyme assays were performed by varying one substrate concentration in the presence of a saturating concentration of the other substrate (see the experimental section). The k_cat values reported are the means calculated from the V_max values obtained for both substrates for each enzyme (the k_cat for AcCS was obtained using OAS and Na₂S; the k_cat for the forward reaction of AcGDH was obtained using hydroxypyruvate and NADPH, whereas that for the reverse reaction was obtained using glycerate and NADP⁺; the k_cat for AcSPAT was obtained using pyruvate and serine). Reactions were performed at 37 °C and results represent means ± SD from triple experiments

ISSN: 1756-3305