Diverse allosteric and catalytic functions of tetrameric d-lactate dehydrogenases from three Gram-negative bacteria

AMB Express

Table 3 Kinetic parameters for pyruvate and NADH ^a

	k_cat(s^-1)	S_0.5(mM)	k_cat/S_0.5(s^-1 mM^-1)	K_i(mM)^b	n _H ^c
Pyruvate
pH 7.0
FNLDH	80 (0.4)	0.34 (0.007)	230 (4)
PALDH	400 (10)	0.10 (0.009)	4,000 (300)	1.8 (0.1)
ECLDH	410 (8)	2.6 (0.09)	160 (2)		1.4 (0.05)
pH 8.0
FNLDH	87 (1)	2.5 (0.06)	35 (0.6)		2.0 (0.1)
PALDH	240 (6)	1.2 (0.1)	200 (10)		0.77 (0.03)
ECLDH	380 (5)	7.6 (0.1)	50 (0.4)		2.2 (0.06)
pH 8.0 + FBP^d
FNLDH	100 (1)	1.7 (0.06)	60 (1)		1.3 (0.04)
PALDH	240 (4)	0.85 (0.04)	280 (6)		0.9 (0.02)
ECLDH	400 (5)	6.5 (0.1)	61 (1)		2.1 (0.09)
pH 8.0 + Mg^+2e
FNLDH	80 (1)	1.2 (0.04)	65 (1)		1.4 (0.05)
PALDH	290 (3)	0.36 (0.01)	820 (20)	20 (0.8)
ECLDH	370 (6)	7.2 (0.2)	51 (1)		1.5 (0.07)
NADH
pH 7.0
FNLDH	98 (0.7)	0.022 (0.0005)	4,400 (80)
PALDH	310 (2)	0.019 (0.0005)	17,000 (400)
ECLDH	640 (20)	0.055 (0.004)	12,000 (500)
pH 8.0
FNLDH	90 (1)	0.036 (0.001)	2,500 (30)		1.6 (0.07)
PALDH	270 (2)	0.035 (0.0007)	7,700 (100)
ECLDH	710 (8)	0.088 (0.002)	8,100 (100)
pH 8.0 + FBP^d
FNLDH	80 (1)	0.028 (0.001)	2,800 (70)		1.5 (0.07)
PALDH	270 (2)	0.035 (0.001)	7,800 (100)
ECLDH	730 (10)	0.092 (0.003)	8,000 (100)
pH 8.0 + Mg^+2e
FNLDH	110 (2)	0.031 (0.001)	3,700 (70)		1.3 (0.05)
PALDH	270 (2)	0.035 (0.0009)	7,700 (100)
ECLDH	690 (9)	0.090 (0.002)	7,700 (100)

^aParameters were determined as described in the `Materials and methods' section, and standard deviations are shown in parentheses. ^bThe inhibition constants (K_i) are shown only for the cases that exhibited significant substrate inhibition. ^cHill coefficients (n_H) are shown only for the cases that exhibited significant cooperativity. ^dThe concentrations of FBP for ECLDH, FNLDH, and PALDH were 5 mM, 10 mM, and 10 mM, respectively. ^eThe concentrations of Mg²⁺ for ECLDH, FNLDH, and PALDH were 2.4 mM, 5 mM, and 5 mM, respectively.