phase peptide synthesis and substrate mimetic ..
First, we prepared a 20-member library via manual SPOT synthesis in which the C-terminal residue of the CVIX sequence was varied to contain any of the 20 proteogenic amino acids. In the first experiment, upon completion of the synthesis, individual spots were excised from the membranes and cut in half. In each case, one half-spot was subjected to enzymatic prenylation with OPP-Far-alkyne followed by click reaction with biotin-azide and visualization with SP-AP. The remaining half-spots were used to confirm that the desired peptides were present on the cellulose filters (). In a secondary experiment for evaluating whether the peptides were prenylated, individual spots were cut in half after completion of the enzymatic prenylation. One half-spot was subjected to screening as described above. Peptides from the other half-spots were photocleaved followed by analysis via MALDI-MS. (). For analysis of the spots subjected to prenylation and visualization with SP-AP, the intensity of the color was normalized to that obtained with CVIQ ( and ). Based on that analysis, eight residues at the X position gave signals comparable to, or greater than A, including E, Q, D, N, G, M, S, and T. Of those, D and E did not show levels of prenylation (ratio of prenylated peptide to unprenylated peptide ) in the secondary MALDI-MS-based screen. Ds-GCVID also showed low reaction rate in a continuous spectrofluorimetric assay (). If streptavidin-horseradish peroxidase (SP-HRP) was used in lieu of SP-AP for screening, peptides containing D and E at the X position manifested much less signal suggesting that those sequences are not efficient substrates. However, the overall level of background staining obtained with SP-HRP was significantly greater than that observed with SP-AP limiting the dynamic range of the the screen. Hence, our preferred procedure is to employ SP-AP as the primary screen and then use SP-HRP in subsequent screening when necessary.
Enzymatic Peptide Synthesis | Request PDF
Post-translational lipidation of a variety of proteins, including Ras, with isoprenoids is essential for normal cellular functions and has important roles in numerous diseases. Consequently there is considerable interest in understanding the enzymes involved in this process and in developing inhibitors that may serve as drugs. Protein prenylation involves the addition of C15 (farnesyl) or C20 (geranylgeranyl) isoprenoids near the C-termini of proteins and is catalyzed by protein prenyltransferases. Following prenylation, additional enzymatic processing including proteolysis and methylation occurrs. To study the substrate specificity of these enzymes, the primary strategy employed to date has involved the synthesis, purification and assaying of individual peptides., While that strategy has yielded important insights, it limits the extent of sequence space that can be studied due to the large number of possible combinations. Combinatorial approaches provide possible strategies for increasing the number of sequences that can be examined. However, combinatorial studies of protein prenylation present a number of challenges. First, prenylation is a post-translational modification that occurs at the C-terminus of a protein. Since solid phase peptide synthesis is typically performed from the C- to N-terminus, more complex synthetic procedures are required. Next, enzymes involved in prenylation are large (~80 kDa or larger) or membrane-bound limiting their penetration into resins typically employed for solid phase synthesis. Finally, isoprenoids are not intrinsically chromogenic and are sensitive to acidic conditions and electrophiles rendering their their addition to peptides on resin difficult to monitor and their subsequent cleavage and sequence analysis more complex.
Two main drawbacks seriously restrict the synthetic value of proteases as reagents in peptide fragment coupling: (i) native proteolytic activity and, thus, risk of undesired peptide cleavage; (ii) limited enzyme specificities restricting the amino acid residues between which a peptide bond can be formed. While the latter can be overcome by the use of substrate mimetics achieving peptide bond formation at nonspecific ligation sites, the risk of proteolytic cleavage still remains and hinders the wide acceptance of this powerful strategy for peptide coupling. This paper reports on the effect of the trypsin point mutant Asp189Glu on substrate mimetic-mediated reactions. The effect of this mutation on the steady-state hydrolysis of substrate mimetics of the 4-guanidinophenyl ester type and on trypsin-specific Lys- and Arg-containing peptides was investigated. The results were confirmed by enzymatic coupling reactions using substrate mimetics as the acyl donor and specific amino acid-containing peptides as the acyl acceptor. The competition assay verifies the predicted shift in substrate preference from Lys and Arg to the substrate mimetics and, thus, from cleavage to synthesis of peptide bonds. The combination of results obtained qualifies the trypsin mutant D189E as the first substrate mimetic-specific peptide ligase.