The binding of acetaldehyde to the active site of ribonuclease: Alterations in catalytic activity and effects of phosphate

Research output: Contribution to journalArticle

24 Scopus citations

Abstract

Ribonuclease A was reacted with [1-13C, 1.2-14 C] and sodium cyanoborohydride in the presence or absence of 0.2 M phosphate. After several hours of incubation at 4°C (pH 7.4) stable acetaldehyde-RNase adducts were formed, and the extent of their formation was similar regardless of the presence of phosphate. Although the total amount of covalent binding was comparable in the absence or presence of phosphate, this active site ligand prevented the inhibition of enzymatic activity seen in its absence. This protective action of phosphate diminished with progressive ethylation of RNase, indicating that the reversible association of phosphate with the active site lysyl residue was overcome by the irreversible process of reductive ethylation. Modified RNase was analysed using 13C proton decoupled NMR spectroscopy. Peaks arising from the covalent binding of enriched acetaldehyde to free amino groups in the absence of phosphate were as follows: NH2-terminal alpha amino group, 47.3 ppm; bulk ethylation at epsilon amino groups of nonessential lysyl residues, 43.0 ppm; and the epsilon amino group of lysine-41 at the active site, 47.4 ppm. In the spectrum of RNase ethylated in the presence of phosphate, the peak at 47.4 ppm was absent. When RNase was selectively premethylated in the presence of phosphate, to block all but the active site lysyl residues and then ethylated in its absence, the signal at 43.0 ppm was greatly diminished, and that arising from the active site lysyl residue at 47.4 ppm was enhanced. These results indicate that phosphate specifically protected the active site lysine from reaction with acetaldehyde, and that modification of this lysine by acetaldehyde adduct formation resulted in inhibition of catalytic activity.

Original languageEnglish (US)
Pages (from-to)103-112
Number of pages10
JournalAlcohol and Alcoholism
Volume22
Issue number2
StatePublished - Dec 1 1987

    Fingerprint

ASJC Scopus subject areas

  • Medicine (miscellaneous)
  • Toxicology
  • Psychiatry and Mental health

Cite this