Regulation of enzymatic activity by active site fatty acylation. A new role for long chain fatty acid acylation of proteins

J Biol Chem. 1994 Mar 4;269(9):6498-505.

Abstract

Methylmalonate semialdehyde dehydrogenase (MMSDH) is a mitochondrial enzyme which can be acylated by myristoyl-CoA analogs (Deichaite, I., Berthiaume, L., Peseckis, S. M., Patton, W. F., and Resh, M. D. (1993) J. Biol. Chem. 268, 13788-13747). Here we describe the mechanisms which mediate regulation of the enzymatic activity of bovine MMSDH by long chain fatty acylation. The substrate specificity of the acylation reaction was measured in vitro using purified MMSDH and the coenzyme A derivative of an 125I-labeled long chain fatty acid (13-iodotridecanoate), an analog of myristoyl-CoA. Long chain fatty acyl CoAs (> 8 carbons) were able to inhibit radiolabeling of MMSDH. In order to study the physiological role of the acylation process in vivo, a system using highly purified mitochondria from COS-1 cells overexpressing MMSDH was exploited. MMSDH was shown to be processed properly, targeted to the mitochondrial fraction, and enzymatically active. The extent of fatty acylation of MMSDH as well as of other mitochondrial proteins was correlated with the mitochondrial energy level. Biochemical evidence as well as site-specific mutagenesis of cysteine 319 revealed that this highly conserved active site cysteine of MMSDH was the target of the fatty acylation. Another member of the aldehyde dehydrogenase family, yeast aldehyde dehydrogenase was also covalently modified by [125I]13-iodotridecanoyl-CoA and thereby inactivated. Furthermore, we demonstrate that glutamate dehydrogenase, an enzyme that has been previously shown to be strongly inhibited by palmitoyl-CoA, is fatty acylated by the 125I-labeled myristoyl-CoA analog. Our data suggest that attachment of long chain fatty acids to proteins is a new and potentially widespread type of enzyme regulation mechanism that we denote active site fatty acylation.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acyl Coenzyme A / metabolism*
  • Acyl Coenzyme A / pharmacology
  • Acylation
  • Aldehyde Oxidoreductases / biosynthesis
  • Aldehyde Oxidoreductases / isolation & purification
  • Aldehyde Oxidoreductases / metabolism*
  • Animals
  • Base Sequence
  • Binding Sites
  • Cattle
  • Cell Line
  • Cloning, Molecular
  • Cysteine
  • Glutamate Dehydrogenase / metabolism
  • Iodine Radioisotopes
  • Methionine / metabolism
  • Methylmalonate-Semialdehyde Dehydrogenase (Acylating)
  • Mitochondria / enzymology*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Oligodeoxyribonucleotides
  • Protein Processing, Post-Translational*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Substrate Specificity
  • Transfection

Substances

  • Acyl Coenzyme A
  • Iodine Radioisotopes
  • Oligodeoxyribonucleotides
  • Recombinant Proteins
  • tridecanoyl coenzyme A
  • S-tetradecanoyl-coenzyme A
  • Methionine
  • Aldehyde Oxidoreductases
  • Methylmalonate-Semialdehyde Dehydrogenase (Acylating)
  • Glutamate Dehydrogenase
  • Cysteine