Identification of two essential histidine residues of ribonuclease T2 from Aspergillus oryzae

Eur J Biochem. 1990 Jan 12;187(1):255-62. doi: 10.1111/j.1432-1033.1990.tb15303.x.

Abstract

Ribonuclease (RNase) T2 from Aspergillus oryzae was modified by diethyl pyrocarbonate and iodoacetic acid. RNase T2 was rapidly inactivated by diethyl pyrocarbonate above pH 6.0 and by incorporation of a carboxymethyl group. No inactivation occurred in the presence of 3'AMP. 1H-NMR titration and photo-chemically induced dynamic nuclear polarization experiments demonstrated that two histidine residues were involved in the active site of RNase T2. Furthermore, analysis of inactive carboxymethylated RNase T2 showed that both His53 and His115 were partially modified to yield a total of one mole of N tau-carboxymethylhistidine/mole enzyme. The results indicate that the two histidine residues in the active site of RNase T2 are essential for catalysis and that modification of either His53 or His115 inactivates the enzyme.

MeSH terms

  • Amino Acid Sequence
  • Amino Acids / analysis
  • Aspergillus / enzymology*
  • Aspergillus oryzae / enzymology*
  • Binding Sites
  • Diethyl Pyrocarbonate / pharmacology
  • Endoribonucleases / antagonists & inhibitors
  • Endoribonucleases / metabolism*
  • Histidine*
  • Hydrogen-Ion Concentration
  • Iodoacetates / pharmacology
  • Iodoacetic Acid
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Molecular Sequence Data
  • Peptide Mapping

Substances

  • Amino Acids
  • Iodoacetates
  • Histidine
  • Endoribonucleases
  • ribonuclease T(2)
  • Diethyl Pyrocarbonate
  • Iodoacetic Acid