Molecular Expression and Characterization of Erythroid-Specific 5-Aminolevulinate Synthase Gain-of-Function Mutations Causing X-Linked Protoporphyria

Table 2 Kinetic and thermostability properties of the purified recombinant human wild-type and mutant ALAS2 enzymes.

Parameter^a	Wild-Type	ΔAT	ΔAGTG	AG	p.Q548X	p.F557X
Crude extract SA (units/mg)^b	1,630 ± 156	4,500 ± 764	4,590 ± 2,180	1,540 ± 304	8,470 ± 1,410	1,990 ± 424
Fold-increase	1.0	2.8	2.8	0.94	5.2	1.2
Purified SA (units/mg)^b	81,700	149,000	251,000	77,500	134,000	103,000
Fold-increase	1.0	1.8	3.1	0.95	1.6	1.3
V_maxGly (units/mg)	104,000 ± 1,210	206,000 ± 3,230	339,000 ± 18,300	77,600 ± 573	199,000 ± 68,700	173,000 ± 30,700
K_mGly (mmol/L)	9.3 ± 1.2	13.0 ± 3.2	7.5 ± 0.5	13.5 ± 3.0	12.0 ± 4.3	7.7 ± 3.0
Glyⁿ	0.99 ± 0.02	0.94 ± 0.10	0.95 ± 0.10	0.96 ± 0.10	1.0 ± 0.0	1.0 ± 0.03
V_maxSucCoA (units/mg)	103,000 ± 15,200	161,000 ± 12,500	171,000 ± 13,400	51,300 ± 3,510	204,000 ± 17,900	196,000 ± 6,450
K_mSucCoA (µmol/L)	40.7 ± 6.2	39.8 ± 3.0	35.7 ± 3.3	40.1 ± 4.1	52.4 ± 6.4	36.3 ± 4.1
SucCoAⁿ	1.5 ± 0.1	1.8 ± 0.1	1.8 ± 0.1	1.5 ± 0.1	1.6 ± 0.1	1.7 ± 0.1
K_mPLP (nmol/L)	21.5 ± 5.4	No Activity	6.2 ± 1.0	650 ± 140	5.9 ± 0.5	148 ± 0.5
t_1/2 45°C (min)^c	11.7 ± 3.6	3.8 ± 0.6	6.1 ± 1.5	12.8 ± 4.3	5.5 ± 2.5	4.1 ± 1.2

Gly, glycine; SucCoA, succinyl CoA; n, Hill number.
^aData are means ± SD for n = 3−5 separate experiments. For data with no SD, n = 1.
^bSA, specific activity; measured at the substrate levels as specified in K_m Determinations in Materials and Methods.
^cALAS2 half-life of homogeneous enzyme in 50 mmol/L HEPES, 1 mmol/L DTT, pH 7.4.

ISSN: 1528-3658