The conserved serine-threonine-serine motif of the carnitine acyltransferases is involved in carnitine binding and transition-state stabilization: a site-directed mutagenesis study.

There has been speculation that the carnitine acyltransferase reaction mechanism may involve the formation of an acyl-serine intermediate. A serine-threonine-serine (STS) motif that is conserved throughout the carnitine acyltransferase family, and is present also in the choline acetyltransferases, contains the only two conserved serines. The functional role of this motif in carnitine octanoyltransferase was probed by using a site-directed mutagenesis strategy to generate all seven possible alanine substitutions: single, double and triple mutants. Kinetic analyses of these mutant enzymes demonstrated that the STS motif is not essential for catalysis, thereby excluding an acyl-serine intermediate from the reaction mechanism. The kinetic analyses support, however, substantial roles for the STS motif in carnitine binding and transition-state stabilization.