Kinetic properties of bovine brain protein L-isoaspartyl methyltransferase determined using a synthetic isoaspartyl peptide substrate.
Protein L-isoaspartyl methyltransferase, an enzyme enriched in brain, is implicated in the repair of age-damaged proteins containing atypical, isoaspartyl peptide bonds. We have investigated the kinetics of methylation using a synthetic peptide substrate having the structure Trp-Ala-Gly-Gly-isoAsp-Ala-Ser-Gly-Glu. Double-reciprocal plots of initial velocity versus concentration of S-adenosylmethionine (AdoMet) at different fixed concentrations of peptide gave straight lines converging at a positive 1/v value and a negative 1/AdoMet value. The product S-adenosylhomocysteine (AdoHcy) was a competitive inhibitor towards AdoMet and a linear mixed-type inhibitor towards peptide. These results are consistent with the rapid-equilibrium random sequential bi-bi mechanism previously proposed for the enzyme, but they also reveal the formation of the dead-end, enzyme-peptide-AdoHcy, complex. The rate constants were: Vmax = 32-34 nmol/min/mg, Kpeptide = 7.6-9.4 microM, KAdoMet = 1.9-2.2 microM, alpha = 0.43-0.53, KAdoHcy = 0.08 microM, gamma = 2.9. The interaction factors alpha and gamma indicate that binding of enzyme to peptide increases its affinity for AdoMet and decreases its affinity for AdoHcy. Methylation was linear with time throughout the transfer of 2 mol of methyl groups/mol of enzyme. This absence of burst kinetics suggests that slow release of products cannot explain the low turnover number.
Johnson
B A
BA
School of Biological Sciences, University of California, Irvine 92717-4550.
Aswad
D W
DW
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000031:analogs & derivatives*; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase; D011496:Protein Methyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012435:S-Adenosylhomocysteine / Q000494:pharmacology; D012436:S-Adenosylmethionine / Q000378:metabolism; D014316:Tritium
D010455:Peptides; D014316:Tritium; D001224:Aspartic Acid; D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; D011496:Protein Methyltransferases; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase