Table 1.

Steady‐state kinetic parameters for the H679A and H725A AspH variants. Apparent turnover numbers (${\mathrm{k}}_{\mathrm{cat}}^{\mathrm{app}}$ ), apparent Michaelis constants (${\mathrm{K}}_{\mathrm{m}}^{\mathrm{app}}$ ), and specificity constants (${\mathrm{k}}_{\mathrm{cat}}$ /${\mathrm{K}}_{\mathrm{m}}$ ) of wt, H679A, and H725A His₆‐AspH_315‐758 determined for Fe^II, 2OG, and the hFX‐EGFD1_86‐124‐4Ser substrate peptide.^[a,b]

Entry		AspH variant	${\mathrm{k}}_{\mathrm{cat}}^{\mathrm{app}}$  [s−1]	${\mathrm{K}}_{\mathrm{m}}^{\mathrm{app}}$  [μM]	${\mathrm{k}}_{\mathrm{cat}}$ /${\mathrm{K}}_{\mathrm{m}}$ [mM−1 s−1]
1	FeII[c]	wt	0.24±0.04	7.2±2.6	33.3±13.2
		H679A	0.18±0.01	4.4±0.8	40.9±7.8
		H725A	0.09±0.01	12.3±3.0	7.3±2.0
2	FeII[d]	wt	0.27±0.04	4.3±1.6	62.8±25.2
		H679A	0.19±0.01	2.7±0.5	70.4±13.6
		H725A	0.10±0.01	8.1±1.8	12.3±3.0
3	2OG	wt	0.31±0.03	1.1±0.4	282±106
		H679A	0.18±0.01	109±20	1.7±0.3
		H725A	0.08±0.01	211±65	0.38±0.13
4	hFX‐EGFD186‐124‐4Ser[e]	wt[f]	n.d.	n.d.	n.d.
		H679A	0.18±0.01	1.5±0.3	120±24.9
		H725A	0.10±0.02	1.7±1.1	58.8±39.8

[a] Determined using 0.2 μM AspH variant or 0.1 μM wt AspH by SPE‐MS. Detailed analyses are given in Supporting Figures S4–S6. [b] Mean of three independent runs (n=3; mean ± standard deviation, SD). [c] Without L‐ascorbate (LAA). [d] With LAA. [e] ${\mathrm{K}}_{\mathrm{m}}$ and ${\mathrm{k}}_{\mathrm{cat}}$ values were determined monitoring the hydroxylation of the hFX‐EGFD1_86‐124‐4Ser substrate peptide (Supporting Figure S2a). [f] Apparent substrate inhibition combined with low detectability of the peptide at low concentrations prevented determination.