Abstract
Four hexapeptides of sequence L-Val-L-Tyr-L-Pro-(Asp)-Gly-L-Ala containing D- or L-aspartyl residues in normal or isopeptide linkages have been synthesized by the Merrifield solid-phase method as potential substrates of the erythrocyte protein carboxyl methyltransferase. This enzyme has been shown to catalyze the methylation of D-aspartyl residues in proteins in red blood cell membranes and cytosol. Using a new vapor-phase methanol diffusion assay, we have found that the normal hexapeptides containing either D- or L-aspartyl residues were not substrates for the human erythrocyte methyltransferase. On the other hand, the L-aspartyl isopeptide, in which the glycyl residue was linked in a peptide bond to the beta-carboxyl group of the aspartyl residue, was a substrate for the enzyme with a Km of 6.3 microM and was methylated with a maximal velocity equal to that observed when ovalbumin was used as a methyl acceptor. The enzyme catalyzed the transfer of up to 0.8 mol of methyl groups/mol of this peptide. Of the four synthetic peptides, only the L-isohexapeptide competitively inhibits the methylation of ovalbumin by the erythrocyte enzyme. This peptide also acts as a substrate for both of the purified protein carboxyl methyltransferases I and II which have been previously isolated from bovine brain (Aswad, D. W., and Deight, E. A. (1983) J. Neurochem. 40, 1718-1726). The L-isoaspartyl hexapeptide represents the first defined synthetic substrate for a eucaryotic protein carboxyl methyltransferase. These results demonstrate that these enzymes can not only catalyze the formation of methyl esters at the beta-carboxyl groups of D-aspartyl residues but can also form esters at the alpha-carboxyl groups of isomerized L-aspartyl residues. The implications of these findings for the metabolism of modified proteins are discussed.
Highlights
Four hexapeptides of sequence L-Val-L-Tyr-L-Pro- U ~ U O(1-3) and in uitro [1,4,5,6]
(Asp)-Gly-L-Alacontaining D- or L-aspartyl residues tion is generally substoichiometric, and it appears in normal or isopeptide linkages have been synthesized that only a subpopulation of molecules in a given protein are by the Merrifield solid-phase method as potential sub- capable of functioning as methyl acceptors [4,5,6,7,8,9]
Lisoaspartyl residues in proteins may be derived from L-aspartyl residues
Summary
Peptides were synthesized manually using standard solid-phase procedures [17] with recent modifications [18]. t-BOC' amino acid derivatives were purchased from Vega Biochemicals (Tucson, A Z ) except for t-BOC-L-Asp-P-OBzlester which wasobtained from Sigma and t-BOC-D-Asp-fi-OBzlester from Chemical Dynamics (South Plainfield, NJ). Hydrolysates were evaporated under reduced pressure a t 60 "C and were resuspended in amino acid analyzer sample buffer Peptide (30 nmol) and carboxypeptidase Y (60 pg of protein) were incubated in a buffer of 66.7 mM sodium citrate (pH 6.0) in a final volume of 60 plfor 24 h a t 37 "C These digestions were quenched as described above. The fluorescent aspartic acid derivatives were applied to a Waters Resolve Cl8 column (5 pm, 3.9 X 150 mm) equilibrated and eluted with 46.8 mM sodium acetate (pH 5.90) and 3.2% methanol. The amount of D- and L-isomers was determined from the relative peak heights after correction for the slightly different fluorescent intensities of the two diastereomeric derivatives found using a racemic mixture of aspartic acid
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