A prenylated protein-specific endoprotease in rat liver microsomes that produces a carboxyl-terminal tripeptide.
The maturation of proteins that contain the C-terminal sequence Cys-Ali-Ali-Xaa (where Ali is usually an aliphatic amino acid and Xaa is a number of different amino acids) involves the attachment of a farnesyl or geranylgeranyl group to the cysteine residue, proteolytic removal of the C-terminal three amino acids, and methylation of the prenylated cysteine alpha-carboxyl group. Two prenylated and radiolabeled peptides were prepared in order to detect the proteolysis step(s) in a cell-free system and to determine the reaction products. These peptides are ECB-NPFRQRRFFC(S-geranylgeranyl)AI[3H]L and ECB-C(S-farnesyl)VI[3H]S (ECB is an extended-chain biotin group) which are patterned after the C-termini of geranylgeranylated and farnesylated G protein gamma-subunits. Incubation of these peptides with rat liver microsomes, but not cytosol, results in the production of radiolabeled dipeptides (I[3H]L and I[3H]S) and tripeptides (AI[3H]L and VI[3H]S) as the major products and smaller amounts of amino acids ([3H]L and [3H]S). A multitude of independent experiments shows that the dipeptides are produced from the tripeptides by secondary proteolysis. Although a portion of the [3H]S produced comes directly from ECB-C(S-farnesyl)VI[3H]S, the KM of > 30 microM for this reaction is significantly higher than the KM of 1.1 microM for the production of VI[3H]S from the farnesylated peptide. This suggests that the carboxypeptidase is not part of the pathway for the maturation of prenylated proteins. Nonprenylated peptides at concentrations of 10-100-fold higher than those of the prenylated substrates did not reduce the amount of tripeptide produced.(ABSTRACT TRUNCATED AT 250 WORDS)
Jang
G F
GF
Department of Chemistry, University of Washington, Seattle 98195.
Yokoyama
K
K
Gelb
M H
MH
D000595:Amino Acid Sequence; D000818:Animals; D010450:Endopeptidases / Q000378:metabolism*; D006868:Hydrolysis; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D011480:Protease Inhibitors; D017368:Protein Prenylation; D051381:Rats; D013347:Subcellular Fractions; D013379:Substrate Specificity
D009842:Oligopeptides; D011480:Protease Inhibitors; D010450:Endopeptidases