FUNCTIONAL REQUIREMENTS OF THE ACTIVE-SITE POSITION-185 IN THE HUMAN ENZYME GALACTOSE-1-PHOSPHATE URIDYLYLTRANSFERASE

The active site of galactose-1-phosphate uridylyltransferase (GALT) in cludes a HPH sequence that has been conserved in all species examined from Escherichia coli to humans, The crystal structure of the E. coli enzyme suggests that this proline is important in positioning the acti ve site histidine (His-166) near the substrate, To examine the role of this proline in the homologous human sequence, we have performed satu rating mutagenesis at Pro 185 within human GALT and characterized each resultant mutant enzyme using a yeast expression system, Activity ana lyses in crude lysates indicated that only proline at position 185 pro duced wild type levels of activity, although five other amino acids, A la, Gly, Ser, Gln, and Glu, all produced partially active enzymes, Wes tern blot analyses of the GALT proteins in these lysates demonstrated that abundance varied from 9-118% of wild-type and was independent of activity, All five active mutant proteins were purified and characteri zed with regard to specific activity, apparent K-m for both substrates , and temperature-dependence of activity, Finally, modeling of these m utations onto the conserved E. coli active site structure was performe d, Together, these results provide functional evidence demonstrating t he critical role of Pro-185 in facilitating the transferase reaction.