The amino terminus of PKA catalytic subunit--a site for introduction of posttranslational heterogeneities by deamidation: D-Asp2 and D-isoAsp2 containing isozymes.
Conserved deamidation of PKA catalytic subunit isozymes Calpha and Cbeta--more than 25% at Asn2 in vivo in both cases--has been shown to yield Asp2- and isoAsp2-containing isozymes (Jedrzejewski PT, Girod A, Tholey A, König N, Thullner S, Kinzel V, Bossemeyer D, 1998, Protein Sci 7:457-469). Isoaspartate formation in proteins in vivo is indicative of succinimide intermediates involved in both the initial deamidation reaction as well as the "repair" of isoAsp to Asp by the action of protein L-isoaspartyl (D-aspartyl) O-methyl transferase (PIMT). L-Succinimide is prone to racemization to D-succinimide, which may hydrolyze to D-isoAsp- and D-Asp-containing diastereomers with, respectively, no and poor substrate character for PIMT. To analyze native PKA catalytic subunit from cardiac muscle for these isomers the N-terminal tryptic peptides (T1) of the enzyme were analyzed following procedures refined specifically with a set of corresponding synthetic peptides. The methods combined high resolution high-performance liquid chromatography and a new mass spectrometric procedure for the discrimination between Asp- and isoAsp-residues in peptides (Lehmann et al., 2000). The results demonstrate the occurrence of D-isoAsp- and D-Asp-containing T1 fragments in addition to the L-isomers. The small amount of the L-isoAsp isomer, representing only part of the D-isoAsp isomer, and the relatively large amounts of the L-Asp and D-Asp isomers argues for an effective action of PIMT present in cardiac tissue.
Kinzel
V
V
Department of Pathochemistry, German Cancer Research Center, Heidelberg, Germany. v.kinzel@dkfz-heidelberg.de
König
N
N
Pipkorn
R
R
Bossemeyer
D
D
Lehmann
W D
WD
D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry*; D020134:Catalytic Domain; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry*; D006868:Hydrolysis; D007527:Isoenzymes; D013058:Mass Spectrometry; D008956:Models, Chemical; D009206:Myocardium / Q000201:enzymology; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase; D011496:Protein Methyltransferases / Q000494:pharmacology; D013237:Stereoisomerism; D013388:Succinimides / Q000737:chemistry
D007527:Isoenzymes; D010455:Peptides; D013388:Succinimides; C032620:succinimide; D001224:Aspartic Acid; D011496:Protein Methyltransferases; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase; D017868:Cyclic AMP-Dependent Protein Kinases