Both the in vivo and in vitro deamidation of asparagine residues in proteins and peptides is now well established. 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. The physiological function od deamidation of asparagine residues has been proposed to be a signal for protein degradation in the cell. However, for most proteins known to contain deamidated asparagine residues, little direct evidence supporting this hypothesis is presently available. The present proposal is to continue our studies on serine hydroxymethyltransferase and ovalbumin, which have been documented to have deamidated asparagine residues. 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 primary method of study is to express the cloned cDNA to obtain enzyme which is not deamidated. Site-directed mutagenesis will also be used to make mutant enzyme forms to block the deamidation of each asparagine residue. 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. These studies will use the same mutant proteins and expressed enzyme obtained for the studies in specific aim 1. Enzyme degradation will be studies in Xenopus oocytes which is a new method developed during our last grant period. We have shown that serine hydroxymethyltransferase is rapidly degraded when injected into oocytes and that the first 14 amino acids at the amino terminus, which contains one of the deamidated asparagine resides, play a role in this rapid degradation. The third specific aim is to determine the mechanism and substrate specificity for protein isoaspartyl methyltransferase. The fourth specific aim is to determine the role of higher order structure (secondary, tertiary, and quaternary) in the deamidation of asparagine residues in ovalbumin and a proteolytically nicked form of this protein called plakalbumin. Using the 3-D structure of these proteins and site- directed mutagenesis the role of higher order structure on deamidation of asparagine residues in proteins will be investigated.
