Molecular evolution of B6 enzymes: Binding of pyridoxal-5'-phosphate and Lys41Arg substitution turn ribonuclease A into a model B6 protoenzyme

Table 2 Catalytic activities of recombinant wild-type and mutant RNases.

	k_cat^a (s^-1)	k_obs^b (s^-1)
RNase-PLP	Ribonuclease activity (tRNA)	Racemization (L-Alanine)	Racemization (L-Aspartate)	β-Decarboxylation (L-Aspartate)
Wild-type^c	36 ± 1.0	(6 ± 1.0) × 10^-7	(3 ± 0.5) × 10^-7	(2 ± 0.2) × 10^-7
K7R	35.6 ± 1.0	(6 ± 0.5) × 10^-7	(7 ± 0.7) × 10^-7	< 10^-9d
K41R^c	0.036 ± 0.002	(2 ± 0.5) × 10^-6	(3 ± 0.5) × 10^-7	(4 ± 0.5) × 10^-6
K7A^e	8 ± 0.5	-	-	-
K41A^e	0.0034 ± 0.0002	-	-	-

^aRibonuclease activity was measured as described [41]; for details, see Methods. The ribonuclease activity of the recombinant wild-type and mutant RNases in the absence of PLP was comparable to that of the corresponding RNase-PLP complexes.
^bThe k_obs value denotes the rate of the reaction (μmol·s^-1) under the given conditions per μmol RNase-PLP adduct. PLP-dependent activities were measured at pH 7.5 and 25°C; the concentration of the RNase-PLP adduct was 11 mM; the substrate concentration was 20 mM (for details, see Methods). Results are the mean ± SE of three independent measurements. The rates of the PLP-dependent reactions in the absence of RNase were below the detection limit of 10^-9 s^-1.
^cThese enzymes catalyze the racemization of L-phenylalanine with a rate of (3 ± 0.4) × 10^-7 s^-1.
^dDetection limit for a reaction with the Marfey assay is 10^-9s^-1.
^eThese RNase variants showed low affinity for PLP (see text), their PLP-dependent activities have not been measured.

ISSN: 1471-2091