Spectropolarimetric Studies of Binary and Ternary Complexes of Octopine Dehydrogenase

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Abstract
The optical rotatory dispersion and the circular dichroism of octopine dehydrogenase were studied in the presence and in the absence of NADH and of substrate analogues.Calculation based on the parameter b0 yields for the apoenzyme a helix content of approx. 27%. The presence of β‐structure is suggested by the particular shapes of the spectra and the great difference between the values of the helix content calculated from A193 and A225.In the far‐ultraviolet region there is no change in the optical rotatory dispersion and circular dichroic spectra after addition of NADH alone or NADH +dl‐arginine.The binding of NADH to the apoenzyme induces a positive extrinsic Cotton effect centered around the absorption maximum of the reduced coenzyme. It also produced a broad positive ellipticity band in the same spectral region. This change in the molecular ellipticity was used to calculate the number of binding sites for NADH.The attachment of NADH to the apoenzyme also gives rise to a modification of the circular dichroic spectrum in the region of absorption of aromatic chromophores.The binding of a substrate analogue to the holoenzyme also induces spectral changes which might result from the spatial rearrangement of tyrosine and tryptophan. This fact is discussed in relation to earlier spectrophotometric studies which have also indicated environmental changes of aromatic chromophores due to the formation of ternary complexes.