The optical biosensor technique, based on the surface plasmon resonance (SPR) phenomenon, was used for real-time measurements of the slow conformational transition (isomerization) which occurs in human phenylalanine hydroxylase (hPAH) on the binding/dissociation of l-phenylalanine (l-Phe). The binding to immobilized tetrameric wt-hPAH resulted in a time-dependent increase in the refractive index (up to approx. 3min at 25°C) with an end point of approx. 75RU (resonance units)/(pmolsubunit/mm2). By contrast, the contribution of binding the substrate (165Da) to its catalytic core enzyme [ΔN(1—102)/ΔC(428—452)-hPAH] was only approx. 2RU/(pmolsubunit/mm2). The binding isotherm for tetrameric and dimeric wt-hPAH revealed a [S]0.5-value of 98±7μM (h = 1.0) and 158±11μM, respectively, i.e. for the tetramer it is slightly lower than the value (145±5μM) obtained for the co-operative binding (h = 1.6±0.4) of l-Phe as measured by the change in intrinsic tryptophan fluorescence. The responses obtained by SPR and intrinsic tryptophan fluorescence are both considered to be related to the slow reversible conformational transition which occurs in the enzyme upon l-Phe binding, i.e. by the transition from a low-activity state ('T-state') to a relaxed high-activity state ('R-state') characteristic of this hysteretic enzyme, however, the two methods reflect different elements of the transition. Studies on the N- and C-terminal truncated forms revealed that the N-terminal regulatory domain (residues 1—117) plus catalytic domain (residues 118—411) were required for the full signal amplitude of the SPR response. Both the on- and off-rates for the conformational transition were biphasic, which is interpreted in terms of a difference in the energy barrier and the rate by which the two domains (catalytic and regulatory) undergo a conformational change. The substrate analogue 3-(2-thienyl)-l-alanine revealed an SPR response comparable with that of l-Phe on binding to wild-type hPAH.
Resolution of the lifetimes and correlation times of the intrinsic tryptophan fluorescence of human hemoglobin solutions using 2 GHz frequency-domain fluorometry.
