Novel Inhibition of Porcine Pepsin by a Substituted Piperidine
Pepsin inhibition by 3-alkoxy-4-arylpiperidine (substituted piperidine; (3 R ,4 R )-3-(4-bromobenzyloxy)-4-[4-(2-naphthalen-1-yl-2-oxo-ethoxy)phenyl]piperidine) has been studied using steady-state kinetic and pre-equilibrium binding methods. Data were compared with pepstatin A, a well known competit...
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| Published in: | The Journal of biological chemistry 2002-08, Vol.277 (32), p.28677-28682 |
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| Main Authors: | , , , , , , , |
| Format: | Article |
| Language: | eng |
| Online Access: | Get full text |
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| Summary: | Pepsin inhibition by 3-alkoxy-4-arylpiperidine (substituted piperidine; (3 R ,4 R )-3-(4-bromobenzyloxy)-4-[4-(2-naphthalen-1-yl-2-oxo-ethoxy)phenyl]piperidine) has been studied using steady-state kinetic
and pre-equilibrium binding methods. Data were compared with pepstatin A, a well known competitive inhibitor of pepsin. Steady-state
analysis reveals that the substituted piperidine likewise behaves as a competitive inhibitor. Pre-equilibrium binding studies
indicate that the substituted piperidine can displace a fluorescently labeled statine inhibitor from the enzyme active site.
Simulation of the stopped-flow fluorescence transients provided estimates of the K
d values of 1.4 ± 0.2 μ m and 39 ± 2 n m for the piperidine and the fluorescently labeled statine, respectively. The effects of combinations of these two inhibitors
resulted in a series of parallel lines when plotted by the method of Yonetani and Theorell (Yonetani, T., and Theorell, H.
(1964) Arch. Biochem. Biophys. 106, 234â251), suggesting that the two inhibitors bind in a mutually exclusive fashion to pepsin. Fitting of the entire data
set to the appropriate equation yielded an α factor of 8 ± 1. The magnitude of this factor (â > α > 1) can be explained by
a conformational distinction between the enzyme species that bind each inhibitor. The effects of pH on the inhibition constants
for pepstatin A and the substituted piperidine also suggest that the inhibitors bind to distinct conformational forms of the
enzyme. No inhibition by the piperidine was observed at acidic pH, while pepstatin A inhibition is maximal at low pH values.
Inhibition by the piperidine was maximal when a group with p K 4.8 ± 0.2 was deprotonated and another group with p K 5.9 ± 0.2 was protonated. Most likely these two groups are the catalytic aspartates with perturbed ionization properties
as a result of a significant and unique conformational change. Taken together, these data suggest that the enzyme can readily
interconvert between two conformers, one capable of binding substrate and pepstatin A and the other capable of binding the
substituted piperidine. |
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| ISSN: | 0021-9258 1083-351X |