Related Experiment Videos
Purification and properties of thioltransferase
The Journal of Biological Chemistry
|January 25, 1986
Summary
This study purified thioltransferase, revealing its cysteine-dependent active center and proposing a catalytic mechanism for thiol-disulfide oxidoreduction. The enzyme shows sensitivity to alkylating reagents and substrate-dependent activity kinetics.
Area of Science:
- Biochemistry
- Enzymology
- Protein Chemistry
Background:
- Thioltransferase, previously identified, required purification for detailed characterization.
- Existing purification methods were suboptimal in terms of speed and yield.
Purpose of the Study:
- To purify thioltransferase to homogeneity.
- To characterize its biochemical properties, including molecular weight, pI, and amino acid composition.
- To elucidate its catalytic mechanism, particularly its role in thiol-disulfide oxidoreduction.
Main Methods:
- Purification using modified preparative procedures.
- Homogeneity assessment via SDS-PAGE and isoelectric focusing.
- Enzyme kinetics studies including pH optimum, substrate concentration effects, and inactivation assays with iodoacetamide.
Main Results:
- Successfully purified thioltransferase with a molecular weight of 11,700 and pI of 8.8.
- Amino acid composition is similar to calf thymus glutaredoxin.
- Optimal activity at pH 8.5 with sigmoidal kinetics for S-sulfocysteine and 2-hydroxyethyl disulfide.
- Demonstrated sensitivity to sulfhydryl alkylating reagents and substrate-mediated protection against inactivation.
Conclusions:
- The active center of thioltransferase is cysteine-dependent.
- Substrates likely form mixed disulfides with the enzyme during catalysis.
- A model for the thiol-disulfide oxidoreduction mechanism was proposed based on inactivation and protection studies.