Brain succinic semialdehyde dehydrogenase: identification of reactive lysyl residues labeled with pyridoxal-5'-phosphate.
An NAD+ dependent succinic semialdehyde dehydrogenase from bovine brain was inactivated by pyridoxal-5'- phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through formation of a Schiff's base with amino groups of the enzyme. After NaBH(4) reduction of the pyridoxal-5'-phosphate inactivated enzyme, it was observed that 3.8 mol phosphopyridoxyl residues were incorporated/enzyme tetramer. The coenzyme, NAD+, protected the enzyme against inactivation by pyridoxal-5'-phosphate. The absorption spectrum of the reduced and dialyzed pyridoxal-5'-phosphate-inactivated enzyme showed a characteristic peak at 325 nm, which was absent in the spectrum of the native enzyme. The fluorescence spectrum of the pyridoxyl enzyme differs completely from that of the native enzyme. After tryptic digestion of the enzyme modified with pyridoxal-5'-phosphate followed by [3H]NaBH4 reduction, a radioactive peptide absorbing at 210 nm was isolated by reverse-phase HPLC. The sequences of the peptide containing the phosphopyridoxyllysine were clearly identical to sequences of other mammalian succinic semialdehyde dehydrogenase brain species including human. It is suggested that the catalytic function of succinic semialdehyde dehydrogenase is modulated by binding of pyridoxal-5'-phosphate to specific Lys(347) residue at or near the coenzyme-binding site of the protein.
Choi
S Y
SY
Department of Genetic Engineering, Division of Life Sciences, Hallym University, Chunchon, Korea. sychoi@sun.hallym.ac.kr
Bahn
J H
JH
Lee
B R
BR
Jeon
S G
SG
Jang
J S
JS
Kim
C K
CK
Jin
L H
LH
Kim
K H
KH
Park
J S
JS
Park
J
J
Cho
S W
SW
D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D004350:Drug Residues / Q000302:isolation & purification; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D020539:Sequence Analysis, Protein; D050644:Succinate-Semialdehyde Dehydrogenase
D000596:Amino Acids; D011732:Pyridoxal Phosphate; D000445:Aldehyde Oxidoreductases; D050644:Succinate-Semialdehyde Dehydrogenase