Abstract：
:The interaction between the beta 2 subunit of tryptophan synthase and the coenzyme pyridoxal 5'-phosphate (PLP) is characterized by induced circular dichroism (CD) in the near-UV (260-285 nm) and in the visible region (320-480 nm, extrinsic Cotton effect). Because of its high mean residue ellipticity ([theta] = 56 deg cm2 dmol-1 for the isolated holo-beta 2 subunit and 102 deg cm2 dmol-1 for the alpha 2-holo-beta 2 complex, respectively) the latter has been used to define different conformational states of the beta 2 dimer via CD titrations. Fitting the obtained binding parameters to the known data from equilibrium dialysis leads to the result that the low-affinity state of the isolated beta 2 subunit shows a 3 times greater rotational strength than the holoenzyme in the high-affinity state. The generation of the final CD amplitude is characterized by a rate constant intermediate between the values for the formation of the internal aldimine and for the regain of enzymatic activity. Interaction of the alpha 2-apo-beta 2 bienzyme complex with the cofactor leads to a hyperbolic binding curve which is apparently free of contributions caused by unspecific PLP binding outside the active center. The determined dissociation constant of 9 x 10(-7) M is in good agreement with the value of 1 x 10(-6) M as obtained by equilibrium dialysis. Binding kinetics reveal a very slow process with a rate constant of 2.6 x 10(-4) s-1, significantly smaller than that for the regain of catalytic activity during reconstitution of the enzyme.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Balk H,Merkl I,Bartholmes Pdoi
10.1021/bi00525a017subject
Has Abstractpub_date
1981-10-27 00:00:00pages
6391-5issue
22eissn
0006-2960issn
1520-4995journal_volume
20pub_type
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