TABLE 2.

Kinetic parameters for the coupled assay of HACN hydrolyase activity with HICDH

Reactions containing 10 μg ml^–1 HICDH, 30 μg ml^–1 HACN holoenzyme, 1 mm NAD⁺, and buffer salts were preincubated at 60 °C before the addition of substrate to initiate the reaction. Hydrolase activity was determined by measuring the rate of increase in UV absorbance at 340 nm caused by the reduction of NAD⁺, as described under “Experimental Procedures.”

Substrate	Km	Vmax	kcat	kcat/Km
	μm	units mg-1	s-1	m-1s-1
cis-Homo1aconitate	25 ± 3	1.4 ± 0.04	1.5	5.9 × 104
cis-Homo2aconitate	20 ± 2	0.93 ± 0.02	1.0	5.0 × 104
cis-Homo3aconitate	46 ± 9	4.7 ± 0.3	5.1	1.1 × 105
cis-Homo4aconitate	NDa	5.8b	ND	ND

^aND, not determined.

^bSpecific activity determined with 0.78 mm cis-homo₄aconitate.