L-cystine inhibits aspartate-beta-semialdehyde dehydrogenase by covalently binding to the essential 135Cys of the enzyme.
Aspartate-beta-semialdehyde dehydrogenase (ASADH) from Escherichia coli is inhibited by L- and D-cystine, and by other cystine derivatives. Enzyme inhibition is quantitatively reversed by addition of dithiothreitol (DTT), dithioerythrytol, beta-mercaptoethanol, di-mercaptopropanol or glutathione to the cystine-inactivated enzyme. Cystine labeling of the enzyme is a pH dependent process and is optimal at pH values ranging from 7.0 to 7.5. Both the cysteine incorporation profile and the inactivation curve of the enzyme as a function of pH suggest that a group(s) with pK(a) of 8.5 could be involved in cystine binding. Stoichiometry of the inactivation reaction indicates that one cysteine residue from the enzyme subunit is reactive against cystine, as found by direct incorporation of radioactive cystine into the enzyme and by free-thiol titration of the enzyme with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) before and after the cystine treatment. One mole of cysteine is released from each mol of cystine after reaction with the enzyme. ASA, NADP and NADPH did not prevent cystine inhibition. The [35S]cysteine-labelled enzyme can be visualized after electrophoresis in polyacrylamide gels and further detection by autoradiography. After pepsin treatment of the [35S]cysteine-inactivated enzyme, a main radioactive peptide was isolated by HPLC. The amino acid sequence of this peptide was determined as FVGGN(Cys)(2)TVSL, thus demonstrating that the essential 135Cys is the amino acid residue modified by the treatment with cystine.
Alvarez
Emilio
E
GlaxoSmithKline Pharmaceuticals, Centro de Investigación Básica, Parque Tecnológico de Madrid, Santiago Grisolía, 4, Tres Cantos, 28760 Madrid, Spain. emilio.alvarez@gsk.com
Ramón
Fernando
F
Magán
Carmen
C
Díez
Emilio
E
D001223:Aspartate-Semialdehyde Dehydrogenase / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000737:chemistry; D001345:Autoradiography; D003553:Cystine / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D010434:Pepsin A; D010446:Peptide Fragments / Q000032:analysis; D013462:Sulfur Radioisotopes
D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D013462:Sulfur Radioisotopes; D003553:Cystine; D001223:Aspartate-Semialdehyde Dehydrogenase; D010434:Pepsin A