Use of Surface Plasmon Resonance for Real-Time Measurements of the Global Conformational Transition in Human Phenylalanine Hydroxylase in Response to Substrate Binding and Catalytic Activation
✍ Scribed by Torgeir Flatmark; Anne Jorunn Stokka; Sissel V. Berge
- Publisher
- Elsevier Science
- Year
- 2001
- Tongue
- English
- Weight
- 93 KB
- Volume
- 294
- Category
- Article
- ISSN
- 0003-2697
No coin nor oath required. For personal study only.
✦ Synopsis
In the present study the optical biosensor technique, based on the surface plasmon resonance (SPR) phenomenon, was used for real-time measurements of the reversible binding of the pterin cofactor (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) and l-phenylalanine (l-Phe) to human phenylalanine hydroxylase (hPAH). When BH(4) (241 Da) was injected over the sensor chip with immobilized tetrameric wt-hPAH a positive DeltaRU response was observed with a square-wave type of sensorgram and a saturable response (about 25 RU/(pmol subunit/mm(2)) with a S value of 5.6 +/- 0.8 microM for the pterin cofactor. The rapid on-and-off rates were, however, not possible to determine. By contrast, when l-Phe (165 Da) was injected a time-dependent increase in RU (up to about 3 min) and a much higher saturable DeltaRU response (about 75 RU/(pmol subunit/mm(2)) at 2 mM l-Phe) than expected (i.e., <5 RU/(pmol subunit/mm(2))) from the low molecular mass of l-Phe were observed in the sensorgram. The half-time for the on-and-off rates were 6 +/- 2 and 9 +/- 1 s, respectively, at 2 mM l-Phe. The steady-state (apparent equilibrium) response revealed a hyperbolic concentration dependence with a S value of 98 +/- 7 microM. The S values of both pterin cofactor and l-Phe were lower than those determined by steady-state enzyme kinetic analysis. Evidence is presented that the DeltaRU response to l-Phe is accounted for by the global conformational transition which occurs in the enzyme upon l-Phe binding, i.e., by the slow reversible transition from a low activity state ("T"-state) to a high activity state ("R"-state) characteristic of this hysteretic enzyme.