Abstract:
:An improved purification procedure for the alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli has been developed. It consists of DEAE-Sephacel chromatography, followed by hydrophobic chromatography on Sepharose CL 4B, and leads to material with a higher specific activity than reported previously. Inhibition studies, equilibrium dialysis, and spectrophotometric titration were used to study the binding both of pyridoxal phosphate analogues and of bisubstrate analogues. Pyridoxine 5'-phospate and N-phosphopyridoxyl-L-serine bind to the enzyme, but pyridoxamine 5'-phoshate and N-phosphopridoxyl-L-alanine do not. N-Phosphopyridoxyl-L-tryptophan is bound only weakly, although L-tyrptophan binds strongly to the alpha 2 holo beta 2 complex. It is likely that either differences is protonation or in geometry are responsible for the low affinity of the bisubstrate analogues in comparison to that of the external aldimines of either L-serine or L-tyrptophan with pyridoxal 5'-phosphate. As previously found with pyridoxal 5'-phosphate, pyridoxine 5'-phosphate, and N-phosphopryidoxyl-L-serine bind noncooperatively to two identical binding sites in the alpha 2 apo beta 2 complex. The same ligands bind with positive cooperatively to two binding sites in the apo beta 2 subunit. Because the analogues mimic the binding behavior of pyridoxal 5'-phosphate to both proteins, the internal aldimine of pyridoxal 5'-phosphate to the lysine amino group contributes only to the strength of that binding. The nickel apo beta 2 subunit, which is produced by limited proteolysis with trypsin, binds pyroxine 5'-phosphate noncooperatively to two identical sites. Therefore, the loop of polypeptide chain connecting the two autonomous domains of folding must be intact for enzyme activity, for the binding of the alpha subunit, and for cooperative binding of pyridoxine 5'-phosphate.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Tschopp J,Kirschner Kdoi
10.1021/bi00560a020subject
Has Abstractpub_date
1980-09-16 00:00:00pages
4514-21issue
19eissn
0006-2960issn
1520-4995journal_volume
19pub_type
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