Deamidation of asparagine residues in a recombinant serine hydroxymethyltransferase

Serine hydroxymethyltransferase purified from rabbit liver cytosol has at l east two Asn residues (Asn(5) and Asn(220)) that are 67 and 30% deamidated, respectively. Asn(5) is deamidated equally to Asp and isoAsp, while Asn(22 0) is deamidated only to isoAsp, To determine the effect of these Asn deami dations on enzyme activity and stability a recombinant rabbit liver cytosol ic serine hydroxymethyltransferase was expressed in Escherichia coli over a 5-h period. About 90% of the recombinant enzyme could be isolated with the two Asn residues in a nondeamidated form, Compared with the enzyme isolate d from liver the recombinant enzyme had a 35% increase in catalytic activit y but exhibited no significant changes in either affinity for substrates or stability. Introduction of Asp residues for either Asn(5) or Asn(220) did not significantly alter activity or stability of the mutant forms. In vitro incubation of the recombinant enzyme at 37 degrees C and pH 7.3 resulted i n the rapid deamidation of Asn(5) to both Asp and isoAsp with a t(1/2) of 5 0-70 h, which is comparable to the rate found with small flexible peptides containing the same sequence. The t(1/2) for deamidation of Asn(220) was at least 200 h, This residue may become deamidated only after some unfolding of the enzyme. The rates for deamidation of Asn(5) and Asn(220) are consist ent with the structural environment of the two Asn residues in the native e nzyme. There are also at least two additional deamidation events that occur during prolonged incubation of the recombinant enzyme. (C) 1999 Academic P ress.