Mimicking the active site of protein disulfide-isomerase by substitution of proline 34 in Escherichia coli thioredoxin.
To mimic the active sites (Trp-Cys-Gly-His-Cys) contained in two thioredoxin-like domains of the eukaryotic enzyme protein disulfide-isomerase (PDI, EC 5.3.4.1), the Pro-34 residue of Escherichia coli thioredoxin (Trx) was replaced by His using site-directed mutagenesis. The mutant P34H Trx was isolated in high yield and was stable. The equilibrium between Trx and NADPH in the thioredoxin reductase (TR)-catalyzed reaction revealed that the redox potential (E'o) or P34H Trx at pH 7.0 was -235 mV as compared with -270 mV for wild type (wt) Trx. The higher E'o value made P34H Trx more similar to PDI and contributed to prominent changes in Trx functions, e.g. improved activity with TR and slower reduction of protein disulfides. Compared to wt Trx, the P34H oxidized Trx was about twice as good a substrate for TR from E. coli and four times as efficient with calf thymus TR. A novel fluorimetric assay permitted direct recording of the reaction between insulin disulfide(s) and reduced Trx. At pH 8 and 15 degrees C, second-order rate constants for wt Trx of 2 x 10(4) M-1 s-1 and for P34H Trx of 3 x 10(3) M-1 s-1 were obtained, and a different equilibrium was observed consistent with differences in E'o values. Also when the reduction mechanism of insulin was examined using NADPH and TR, P34H Trx behaved differently from wt Trx or PDI. P34H Trx may be useful as an analogue of PDI for disulfide formation in vivo and in vitro.
Krause
G
G
Department of Physiological Chemistry, Karolinska Institute, Stockholm, Sweden.
Lundström
J
J
Barea
J L
JL
Pueyo de la Cuesta
C
C
Holmgren
A
A
D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism*; D005822:Genetic Vectors; D007535:Isomerases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases; D012689:Sequence Homology, Nucleic Acid; D013050:Spectrometry, Fluorescence; D013879:Thioredoxins / Q000235:genetics*
D013879:Thioredoxins; D007535:Isomerases; D019704:Protein Disulfide-Isomerases