Abstract
Proteinl-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 deadend, enzyme-peptide-AdoHcy, complex. The rate constants were:V max=32–34 nmol/min/mg,K peptide=7.6–9.4 μM,K AdoMet=1.9–2.2 μM, α=0.43–0.53,K AdoHcy=0.08 μM, γ=2.9. The interaction factors α and γ 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.
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Johnson, B.A., Aswad, D.W. Kinetic properties of bovine brain proteinl-isoaspartyl methyltransferase determined using a synthetic isoaspartyl peptide substrate. Neurochem Res 18, 87–94 (1993). https://doi.org/10.1007/BF00966926
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DOI: https://doi.org/10.1007/BF00966926