Structure and enzymatic functions of thioredoxin refolded by complementation of two tryptic peptide fragments.

Abstract:

:The physicochemical and catalytic properties of thioredoxin-T' are described. This complemented protein structure consists of a 1:1 complex between the inactive fragments thioredoxin-T-(1--73) and thioredoxin T-(74--108). These are generated by selective trypsin cleavage at Arg-73 in lysine-modified and denatured Escherichia coli thioredoxin. Thioredoxin-T' was a slowly formed but stable complex with an apparent KD below 10(-8) M. The tryptophan fluorescence spectrum and the CD spectrum were very similar to those of native thioredoxin; some conformational differences were detected by gel chromatography and radioimmunoassay. Thioredoxin-T'-S2 was a substrate for NADPH and thioredoxin reductase and had 1--2% of the activity of native thioredoxin. This low relative activity was the result of a major increase in the Km value. Thioredoxin-(SH)2 was a hydrogen donor for E. coli ribonucleotide reductase with about 3% relative activity. These results for thioredoxin-T' are correlated with the known three-dimensional structure of thioredoxin. The microenvironment around Arg-73 that is close to the active disulfide appears to be of critical importance for the interactions of thioredoxin with thioredoxin reductase and ribonucleotide reductase.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Slabý I,Holmgren A

doi

10.1021/bi00592a010

subject

Has Abstract

pub_date

1979-12-11 00:00:00

pages

5584-91

issue

25

eissn

0006-2960

issn

1520-4995

journal_volume

18

pub_type

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