title: Conformational change of glyceraldehyde-3-phosphate dehydrogenase induced by acetylleucine chloromethyl ketone is followed by unique enzymatic degradation.
abstract: We have previously reported that acetylleucine chloromethyl ketone (ALCK), an inhibitor of acylpeptidehydrolase, induces the inhibition and degradation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the U937 cell extract. In the present study, the process of ALCK-induced GAPDH degradation was investigated. A kinetic study revealed that GAPDH was irreversibly inhibited by ALCK. ALCK treatment induced a change in the signal intensity of GAPDH in the near-UV region of the circular dichroism (CD) spectrum, and the fluorescence intensity of GAPDH at 330 nm increased to about 10% when excited at 280 nm, suggesting that a significant conformational change of GAPDH was induced by ALCK. When the U937 cell extract was incubated with ALCK and the products were separated by SDS-polyacrylamide gel electrophoresis (PAGE), a 23-kDa fragment from GAPDH was detected by Western blotting using anti-GAPDH serum. When ALCK-treated GAPDH was incubated with protease fractions from the U937 cell extract, a 17-kDa fragment was also detected. Sequence analysis showed that the N-terminal amino acid sequence of the 23-kDa fragment was GKVKVG and that of 17-kDa fragment was RDGRGAL. Therefore, ALCK-modified GAPDH is deduced to be digested at the peptide bond Trp(195)-Arg(196). The protease activity liberating a 23-kDa fragment from ALCK-treated GAPDH was effective under the basic condition. Results suggested that ALCK binds to GAPDH to modulate the conformation of enzyme, which is susceptible to chymotrypsin-like protease activity.
authors.affiliation: Department of Hygienic Chemistry, Showa Pharmaceutical University, Tokyo, Japan. mitsune@ac.shoyaku.ac.jp
authors.forename: Mitsune
authors.initials: M
authors.lastname: Yamaguchi
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Tsuchiya
authors.forename: Kayoko
authors.initials: K
authors.lastname: Hishinuma
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Chikuma
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hojo
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000378:metabolism*; D000596:Amino Acids / Q000737:chemistry; D015153:Blotting, Western; D002457:Cell Extracts; D053818:Chymases; D002942:Circular Dichroism / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004912:Erythrocytes / Q000201:enzymology; D005453:Fluorescence; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects*; D012697:Serine Endopeptidases / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D020298:U937 Cells
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D000596:Amino Acids; D002457:Cell Extracts; D010446:Peptide Fragments; C121811:acetylleucine chloromethyl ketone; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D012697:Serine Endopeptidases; D053818:Chymases