Deamidation of Asparagine Residues in a Recombinant Serine Hydroxymethyltransferase

Abstract

Serine hydroxymethyltransferase purified from rabbit liver cytosol has at least two Asn residues (Asn5 and Asn220) that are 67 and 30% deamidated, respectively. Asn5 is deamidated equally to Asp and isoAsp, while Asn220 is deamidated only to isoAsp. To determine the effect of these Asn deamidations on enzyme activity and stability a recombinant rabbit liver cytosolic 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 isolated from liver the recombinant enzyme had a 35% increase in catalytic activity but exhibited no significant changes in either affinity for substrates or stability. Introduction of Asp residues for either Asn5 or Asn220 did not significantly alter activity or stability of the mutant forms. In vitro incubation of the recombinant enzyme at 37°C and pH 7.3 resulted in the rapid deamidation of Asn5 to both Asp and isoAsp with a t1/2 of 50–70 h, which is comparable to the rate found with small flexible peptides containing the same sequence. The t1/2 for deamidation of Asn220 was at least 200 h. This residue may become deamidated only after some unfolding of the enzyme. The rates for deamidation of Asn5 and Asn220 are consistent with the structural environment of the two Asn residues in the native enzyme. There are also at least two additional deamidation events that occur during prolonged incubation of the recombinant enzyme.