Human thioredoxin reductase from HeLa cells: selective alkylation of selenocysteine in the protein inhibits enzyme activity and reduction with NADPH influences affinity to heparin

Abstract

Human thioredoxin reductase (TR) contains selenocysteine (Secys) in a redox center [cysteine (Cys)-497,Secys-498] near the C-terminus. The essential role of Secys in TR isolated from HeLa cells was demonstrated by the alkylation studies. Reaction of native NADPH reduced enzyme with bromoacetate at pH 6.5 inhibited enzyme activity 99%. Of the incorporated carboxymethyl (CM) group, 1.1 per subunit, >90% was in CM-Secys-498. Alkylation at pH 8 increased the stoichiometry to 1.6 per subunit with additional modification of the Cys-59, Cys-64 disulfide center. A minor tryptic peptide containing both CM-Cys-497 and CM-Secys-498 was isolated from enzyme alkylated at pH 6.5 or at pH 8. Preparations of TR isolated from HeLa cells grown in a fermentor under high aeration contained selenium-deficient enzyme species that had 50% lower activity. Decreasing oxygen to an optimal level increased cell yield, and fully active TR containing one Se per subunit was present. Reduction of fully active enzyme with tris-(2-carboxyethyl) phosphine converted it from a low to a high heparin affinity form. The tris-(2-carboxyethyl) phosphine-reduced enzyme was oxygen-sensitive and lost selenium and catalytic activity unless maintained under strictly anaerobic conditions. This enzyme could be converted to an oxygen-insensitive species by addition of NADPH, indicating that bound pyridine nucleotide is important for enzyme stability. An induced enzyme conformation in which the essential Secys is shielded from oxidative damage could explain these effects.

Figure 1

TCEP reduction of a low heparin affinity form of TR converts it to a high heparin affinity form. Low heparin affinity TR (≈70 μg) was rechromatographed on a heparin HPLC column in buffer A (dotted line). Another 70-μg sample was reduced with 1 mM TCEP in buffer A and was chromatographed on the same column equilibrated with 1 mM TCEP in buffer A (solid line). In both cases, after a 30 min wash with the application buffer, a linear KCl gradient (0–1 M) in the same buffer was applied for 20 min at a flow rate of 0.5 ml/min.

Figure 2

DTNB reduction by TR as a function of pH. Potassium phosphate buffers (100 mM) of the indicated pH were used.

Figure 3

Reversed-phase HPLC separation of a tryptic digest of bromo[1-¹⁴C]acetate alkylated ⁷⁵Se-labeled TR from HeLa cells. Conditions for alkylation, digestion with trypsin, and HPLC separation are described in Materials and Methods. Solid circles, ¹⁴C radioactivity; open circles, ⁷⁵Se radioactivity. (A) Edman degradation profile of ¹⁴C [solid bars] and ⁷⁵Se [open bars] recovered at each sequencer cycle from the major tryptic peptide eluted in fractions 96–97. (B) Profile as above from a minor peptide eluted in fractions 91–92. The amino acid identified in each cycle of A and B, shown in the line between the inserts, is the same as the predicted sequence of human placenta TR (5).