HETERODIMER FORMATION AND ACTIVITY IN THE HUMAN ENZYME GALACTOSE-1-PHOSPHATE URIDYLYLTRANSFERASE

One of the fundamental questions concerning expression and function of dimeric enzymes involves the impact of naturally occurring mutations on subunit assembly and heterodimer activity. This question is of part icular interest for the human enzyme galactose-1-phosphate uridylyltra nsferase (GALT), impairment of which results in the inherited metaboli c disorder galactosemia, because many if not most patients studied to date are compound heterozygotes rather than true molecular homozygotes . Furthermore, the broad range of phenotypic severity observed in thes e patients raises the possibility that allelic combination, not just a llelic constitution, may play some role in determining outcome, in the work described herein, we have selected tao distinct naturally occurr ing null mutations of GALT, Q188R and R333W, and asked the questions ( i) what are the impacts of these mutations on subunit assembly, and (i i) if heterodimers do form, are they active? To answer these questions , we have established a yeast system for the coexpression of epitope-t agged alleles of human GALT and investigated both the extent of specif ic GALT subunit interactions and the activity of defined heterodimer p ools. We have found that both homodimers and heterodimers do form invo lving each of the mutant subunits tested and that both heterodimer poo ls retain substantial enzymatic activity. These results are significan t not only in terms of their implications for furthering our understan ding of galactosemia and GALT holoenzyme structure-function relationsh ips but also because the system described may serve as a model for sim ilar studies of other complexes composed of multiple subunits.