Glutathione Reductase from Human Erythrocytes: The Sequences of the NADPH Domain and of the Interface Domain

Abstract

Sequence analysis of the NADPH domain (residues 158 – 293) and of the interface domain (365 – 478) was based on 12 CNBr fragments, which were isolated using ion‐exchange chromatography and paper methods. Fragments with more than 15 residues were digested further with trypsin and chymotrypsin. The isolated peptides were sequenced by automated solid‐phase Edman degradation. All sequenced peptides were ordered and overlapped by computerized comparisons with a complete sequence guessed from the electron density map of the protein. In the case of short CNBr fragments, this alignment was confirmed by the sequence analysis of protein fragments resulting from incomplete CNBr cleavage. In the NADPH domain, residue 197, which is involved in an induced‐fit mechanism, was identified as a tyrosine. The structure of the NADPH domain is probably homologous with the NAD domain of lipoamide dehydrogenase and with the FAD domain of several proteins, but not with NADPH domains of known chainfold in other proteins. The paper completes the sequence analysis of glutathione reductase so that the enzyme is now known in atomic detail. The numbering scheme of the chemically determined sequence will be used henceforth in crystallo‐ graphic studies also. As inferred from the sequence data each of the two identical chains contains 478 amino acid residues, the composition being Cys 10, Asp 21, Asn 17, Thr 31, Ser 31, Glu 29, Gln 11, Pro 24, Gly 43, Ala 42, Val 44, Met 15, He 29, Leu 34, Tyr 13, Phe 14, Lys 34, His 16, Arg l7, and Trp 3. From these data an M r of 2 × 51600 was calculated for the FAD‐free apoenzyme and an M r of 2 × 52400 for the holoenzyme.