Peptides isolated from Enterobacter nuclease as potential polyamine binding sites

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Enterobacter nuclease is an enzyme that cleaves RNA.
• Polyamines are small organic molecules with positive charges that interact with nucleic acids and proteins.
• The effect of polyamines on Enterobacter nuclease activity was previously unknown.

Purpose of the Study:

• To investigate the influence of polyamines (spermidine, spermine, putrescine) on the hydrolytic activity of Enterobacter nuclease.
• To explore the mechanism by which polyamines affect enzyme inhibition by ordered polynucleotides.
• To identify and characterize polyamine-bound peptides derived from Enterobacter nuclease.

Main Methods:

• Enzyme assays were performed to measure the hydrolytic activity of Enterobacter nuclease in the presence of various polyamines.
• Inhibition reversal studies were conducted using ordered polynucleotides and polyamines.
• Tryptic digests of poly(G)-bound nuclease were treated with spermidine, followed by purification using ion-exchange chromatography.

Main Results:

• Polyamines (spermidine, spermine, putrescine) significantly enhance the RNA-cleaving activity of Enterobacter nuclease.
• Spermidine and spermine reversed the inhibition of the enzyme caused by ordered polynucleotides, suggesting displacement from the enzyme surface.
• Three spermidine-bound peptides of varying lengths and amino acid compositions were isolated, with spermidine-to-peptide binding ratios ranging from 1:1 to 3:1.

Conclusions:

• Polyamines are important modulators of Enterobacter nuclease activity, enhancing its catalytic function.
• The interaction of polyamines with the enzyme or its substrates plays a role in regulating nuclease activity.
• Specific polyamine-binding peptides were identified, providing insights into polyamine-peptide interactions at a molecular level.