Pyridoxal 5'-phosphate binding of a recombinant rat serine: pyruvate/alanine:glyoxylate aminotransferase.
Serine: pyruvate/alanine: glyoxylate aminotransferase in the liver is a class IV amino-transferase. The present study was undertaken to characterize the pyridoxal 5'-phosphate (PLP) binding to a recombinant rat serine: pyruvate/alanine: glyoxylate aminotransferase (SPT10), which is a homodimer of 44.4 kDa subunits. Purified SPT10 exhibited absorption maxima at approximately 330 nm in addition to a 278 nm protein peak and a approximately 420 nm peak of PLP bound via Schiff base, and contained 0.56-0.69 mol of PLP per mol of subunit. Apo-SPT10 without measurable bound PLP did not exhibit the absorbance at approximately 420 nm, but still showed the approximately 330 nm peak. Upon reconstitution, 0.73-0.79 mol of PLP per mol of subunit was bound to apo-SPT10 with an apparent Kd of approximately 0.1 microM, resulting in a holo-SPT10 preparation whose specific activity and A approximately 420/A approximately 330 absorbance ratio were higher than those of the original SPT10. On SDS/PAGE of BrCN-cleavage peptides of NaBH4-reduced SPT10, 22-23 kDa fragments migrated as a pair of bands. On amino acid sequencing, the approximately 22 and approximately 23 kDa pair gave the same sequence, except that Lys was released only from the approximately 22 kDa band material in the cycle corresponding to Lys209. NaB3H4-treated SPT10 also migrated as a pair of 44-45 kDa bands and 3H was incorporated only into the approximately 45 kDa band. It appears that SPT10 has the capacity to bind 1 mol of PLP to Lys209 of every subunit, but usually binds less PLP in a Schiff base structure, probably due to the presence of a 330 nm-absorbing chromophore.
Ishikawa
K
K
First Department of Biochemistry, Hamamatsu University School of Medicine, Shizuoka.
Kaneko
E
E
Ichiyama
A
A
D000410:Alanine Transaminase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001051:Apoenzymes / Q000378:metabolism; D001219:Aspartate Aminotransferases / Q000737:chemistry; D051263:D-Alanine Transaminase; D004926:Escherichia coli / Q000201:enzymology; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012545:Schiff Bases / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013552:Swine; D000637:Transaminases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D001051:Apoenzymes; D011994:Recombinant Proteins; D012545:Schiff Bases; D011732:Pyridoxal Phosphate; D000637:Transaminases; D001219:Aspartate Aminotransferases; D000410:Alanine Transaminase; D051263:D-Alanine Transaminase; C019635:Alanine-glyoxylate transaminase; C022638:serine-pyruvate aminotransferase; D008239:Lysine