REGULATION OF ENZYMATIC-ACTIVITY BY ACTIVE-SITE FATTY ACYLATION - A NEW ROLE FOR LONG-CHAIN FATTY-ACID ACYLATION OF PROTEINS

Methylmalonate semialdehyde dehydrogenase (MMSDH) is a mitochondrial e nzyme which can be acylated by myristoyl-CoA analogs (Deichaite, I., B erthiaume, L., Peseckis, S. M., Patton, W. F., and Resh, M. D. (1993) J. Biol. Chem. 268, 13788-13747). Here we describe the mechanisms whic h mediate regulation of the enzymatic activity of bovine MMSDH by long chain fatty acylation. The substrate specificity of the acylation rea ction was measured in vitro using purified MMSDH and the coenzyme A de rivative of an I-125-labeled long chain fatty acid (13-iodotridecanoat e), an analog of myristoyl-CoA. Long chain fatty acyl CoAs (>8 carbons ) were able to inhibit radiolabeling of MMSDH. In order to study the p hysiological role of the acylation process in vivo, a system using hig hly purified mitochondria from COS-1 cells overexpressing MMSDH was ex ploited. MMSDH was shown to be processed properly, targeted to the mit ochondrial fraction, and enzymatically active. The extent of fatty acy lation of MMSDH as well as of other mitochondrial proteins was correla ted 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 a cylation. Another member of the aldehyde dehydrogenase family, yeast a ldehyde dehydrogenase was also covalently modified by [I-125]13-iodotr idecanoyl-CoA and thereby inactivated. Furthermore, we demonstrate tha t glutamate dehydrogenase, an enzyme that has been previously shown to be strongly inhibited by palmitoyl-CoA, is fatty acylated by the I-12 5-labeled myristoyl-CoA analog. Our data suggest that attachment of lo ng chain fatty acids to proteins is a new and potentially widespread t ype of enzyme regulation mechanism that we denote active site fatty ac ylation.