Conformational change of glyceraldehyde-3-phosphate dehydrogenase induced by acetylleucine chloromethyl ketone is followed by unique enzymatic degradation.
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.
Yamaguchi
Mitsune
M
Department of Hygienic Chemistry, Showa Pharmaceutical University, Tokyo, Japan. mitsune@ac.shoyaku.ac.jp
Tsuchiya
Yukihiro
Y
Hishinuma
Kayoko
K
Chikuma
Toshiyuki
T
Hojo
Hiroshi
H
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
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