Enzyme degradation will be studies in Xenopus oocytes which is a new method developed during our last grant period. These studies will use the same mutant proteins and expressed enzyme obtained for the studies in specific aim 1. The second specific aim is to determine whether the deamidation of the two asparagine residues in serine hydroxymethyltransferase are signals for enzyme degradation in vivo. Site-directed mutagenesis will also be used to make mutant enzyme forms to block the deamidation of each asparagine residue. The primary method of study is to express the cloned cDNA to obtain enzyme which is not deamidated. The first specific aim is to determine if the in vivo deamidation of two asparagine residues in serine hydroxymethyltransferase occurs by a non- enzymatic, autocatalytic, or enzymatic mechanism. The present proposal is to continue our studies on serine hydroxymethyltransferase and ovalbumin, which have been documented to have deamidated asparagine residues. However, for most proteins known to contain deamidated asparagine residues, little direct evidence supporting this hypothesis is presently available. The physiological function od deamidation of asparagine residues has been proposed to be a signal for protein degradation in the cell. Much work has been done on the mechanism of the deamidation reactions in peptides, but little is known about the mechanism of deamidation in proteins. Both the in vivo and in vitro deamidation of asparagine residues in proteins and peptides is now well established.
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