Synthetic substrate for eukaryotic signal peptidase. Cleavage of a synthetic peptide analog of the precursor region of preproparathyroid hormone.
A synthetic peptide analog of the precursor region of preproparathyroid hormone has been shown to be a specific substrate for hen oviduct signal peptidase. The sequence of the 31-residue peptide is Ser-Ala-Lys-Asp-norleucine (Nle)-Val-Lys-Val-Nle-Ile-Val-Nle-Leu-Ala-Ile-Ala-Phe-Leu-Ala-Arg-Ser-As p-Gly-Lys-Ser-Val-Lys-Lys-Arg-D-Tyr-amide (Caulfield, M. P., Duong, L. T., O'Brien, R., Majzoub, J. A., and Rosenblatt, M. (1988) Mol. Endocrinol. 2, 452-458). This sulfur-free signal peptide analog can be labeled with 125I on the C-terminal D-tyrosine and is cleaved by purified hen oviduct signal peptidase between Gly and Lys, the correct site of cleavage of preproparathyroid hormone in vivo. Amino acid sequence analysis of the cleavage product released 125I at the seventh cycle of Edman degradation, confirming that enzymatic cleavage occurs at the physiological site. Synthetic peptide analogs of the substrate with Lys, Pro, or Asp substituted for Nle-18 were poor substrates for the enzyme and were also poor competitive inhibitors of catalysis, suggesting that modifications at position -18, 12 amino acids from the site of cleavage, directly influence binding by the enzyme. Analysis of the reactivity of signal peptidase with these synthetic peptides provides insight into the cleavage specificity requirements of this eukaryotic signal peptidase.
Caulfield
M P
MP
Department of Biological Research and Molecular Biology, Merck Sharp and Dohme Research Laboratory, West Point, Pennsylvania 19486.
Duong
L T
LT
Baker
R K
RK
Rosenblatt
M
M
Lively
M O
MO
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D010450:Endopeptidases / Q000378:metabolism*; D005260:Female; D007700:Kinetics; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D010057:Oviducts / Q000201:enzymology; D010281:Parathyroid Hormone / Q000138:chemical synthesis*; D010455:Peptides / Q000138:chemical synthesis*; D011498:Protein Precursors / Q000138:chemical synthesis*; D021382:Protein Sorting Signals / Q000378:metabolism; D012697:Serine Endopeptidases; D013379:Substrate Specificity
D008565:Membrane Proteins; D010281:Parathyroid Hormone; D010455:Peptides; D011498:Protein Precursors; D021382:Protein Sorting Signals; C019056:preproparathormone; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase