The Q188R mutation in human galactose-1-phosphate uridylyltransferase acts as a partial dominant negative

A longstanding goal in the fields of molecular genetics and biochemistry has been to explain how naturally occurring mutations associated with human metabolic disease impair activity of the enzymes involved. This goal is particularly complex for enzymes composed of multiple subunits, because single mutations may exert both intra- and intersubunit effects on holoenzyme structure and function. We have previously applied a yeast coexpression system for human galactose-1-phosphate uridylyltransferase, a dimeric enzyme associated with galactosemia, to investigate the impact of naturally occurring mutations on subunit association and holoenzyme function (1). Here we describe the purification and characterization of two heterodimers, R333W/wild type (WT) and Q188R/WT, revealing that although the first exhibits similar to 50% wild-type activity, the second exhibits only similar to 15% wild-type activity. Neither heterodimer varied significantly hom the wild type with regard to apparent K-m for either substrate, although Q188R/WT but not R333W/WT heterodimers demonstrated significantly increased thermal sensitivity relative to the wild-type enzyme. These results demonstrate for the first time a partial dominant negative effect caused by a naturally occurring mutation in human galactose-1-phosphate uridylyltransferase.