JBC, Vol. 250, Issue 19, 7554-7563, Oct, 1975
Substrate-protein interaction in tryptophanase from Bacillus alvei. Kinetic and spectral evaluations
J. D. Fenske and R. D. DeMoss
This investigation studied the substrate protein interaction of the alpha,
beta elimination reaction in tryptophanase (EC 4.1.99.1). The results of
this work are 2-fold. (a) The presence of multiple enzyme sites was found
to be related to the observed kinetic patterns of inhibition. Indole
analogues caused competitive inhibition in the tryptophanase reaction and
noncompetitive inhibition in the dehydratase reaction. Inhibition patterns
of alanine for these activities were reserved. (b) Under some conditions,
compounds which bind presumably at the indole site modified the spectral
and fluorescent characteristics of the enzyme. The addition of anthranilate
to the enzyme resulted in a broad absorption band around 350 nm. This
absorption band was distinct from that formed by alanine addition. Based on
absorption data, both of these compounds could be bound simultaneously. The
optical activity of tryptophanase was reported for the first time. Indole
analogues caused greater conformational alterations in the circular
dichroism spectra than 3-carbon analogues. The calculated anisotrophy
factors, as well as fluorescent quenching data, suggest a more direct
interaction between indole analogues and pyridoxal-P than between 3-carbon
compounds that the coenzyme. It is proposed that the indole site is the
dominant recognition site. The data are consistent with the
three-dimensional aspects of space-filling models of Schiff's bases
evaluated in terms of multiple site binding.
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