Table 1.

Kinetic Parameters for the Reduction of Wild-Type and Mutant TyrH^a

enzyme	reductant	redox potential (mV)	k2b (mM−1 s−1)	klimc (s−1)b	Kdd (mM)
wild-type TyrH	6-methyltetrahydropterin	270e	6.2 ± 0.1	5.9 ± 0.2	0.95 ± 0.10
	tetrahydrobiopterin	270e	2.8 ± 0.1
	5-methyltetrahydrobiopterin	400f	0.11 ± 0.01
	ascorbate	330g	0.035 ± 0.001
	1,4-benzoquinone	99h	0.16 ± 0.01
	glutathione	900i	0.00044 ± 0.00001j
E332A TyrH	6-methyltetrahydropterin		8.1 ± 0.8	9.5 ± 1.5	1.2 ± 0.3
S40E TyrH	6-methyltetrahydropterin		8.7 ± 0.6	7.6 ± 0.5	0.87 ± 0.12
	tetrahydrobiopterin		2.4 ± 0.1

^aConditions: 100 mM Hepes, pH 7.5, 10% glycerol, 0.1 M KCl, 20 °C.

^bk₂ is the second-order rate constant for reduction. Where the reaction exhibited simple second-order kinetics, k₂ was determined from a linear fit of the data. Where the reaction exhibited saturation kinetics, k₂ was determined from a fit to eq 2.

^ck_lim is the first-order rate constant for reduction from a fit of the data to eq 2.

^dK_d is the dissociation constant for the reductant determined from a fit of the data to eq 2.

^eFrom the one-electron redox potential for BH₄ (36), assuming that the change from a dihydroxypropyl group to a methyl group at C6 does not have a significant effect.

^fReference 36.

^gReference 37.

^hReference 38.

ⁱReference 39.

^jThe reduction of TyrH by glutathione was only characterized at a single glutathione concentration. The second-order rate constant for reduction of the wild-type TyrH by glutathione was determined by fitting the data to eq 1 and dividing the observed first-order rate constant by the concentration of glutathione (1 mM).