REPAIR OF SPONTANEOUSLY DEAMIDATED HPR PHOSPHOCARRIER PROTEIN CATALYZED BY THE L-ISOASPARTATE-(D-ASPARTATE) O-METHYLTRANSFERASE

The non-enzymatic deamidation at residues Asn-la and Asn-38 of Escheri chia coli phosphocarrier protein, HPr, and the repair of the resulting L-isoaspartyl (or beta-aspartyl) derivatives, HPr-1 and HPr-2, by rec ombinant human S-adenosylmethionine-dependent L-isoaspartate-(D-aspart ate) O-methyltransferase (EC 2.1.1.77) were investigated. HPr is a com ponent of the bacterial phosphoenolpyruvate:sugar phosphotransferase s ystem that is involved in the concomitant translocation and phosphoryl ation of many hexose sugars. The major products of the deamidation rea ction, L-isoaspartyl (or P-aspartyl) residues at positions 12 and 38, were found to be substrates for the L-isoaspartate-(D-aspartate) O-met hyltransferase, an enzyme active on a wide variety of peptides and pro teins containing these abnormal residues. This enzyme has been shown t o catalyze the first step in a process that can convert L-isoaspartyl residues in peptides to normal L-aspartyl residues. The affinity of a recombinant human methyltransferase for HPr-1, a form deamidated at As n-38, was relatively poor (K-m = 3.6 mM), while a greater affinity was found for HPr-2, a form deamidated at both Asn-12 and Asn-38 (K-m = 1 97 mu M). When HPr-2 was incubated with S-adenosylmethionine and the m ethyltransferase, the bulk of the L-isoaspartyl residues at position 1 2 was converted to L-aspartyl residues, The major by-product was the D -isoaspartyl form. The conversion of L-isoaspartyl residues at positio n 38 to L-aspartyl residues was less complete, reflecting the lower af finity of the methyltransferase for this site. The phosphohydrolysis a ctivity of the repaired form was found to be midway between the form c ontaining only L-aspartyl residues at positions 12 and 38 and the deam idated HPr-2 form.