Novel inhibition of porcine pepsin by a substituted piperidine. Preference for one of the enzyme conformers.
Pepsin inhibition by 3-alkoxy-4-arylpiperidine (substituted piperidine; (3R,4R)-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 microm and 39 +/- 2 nm 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, 234-251), 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 alpha factor of 8 +/- 1. The magnitude of this factor ( infinity > alpha > 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 pK 4.8 +/- 0.2 was deprotonated and another group with pK 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.
Marcinkeviciene
Jovita
J
Department of Chemical Enzymology, Bristol-Myers Squibb Pharmaceutical Company, Wilmington, Delaware 19880-0400, USA. jovita.marcinkeviciene@bms.com
Kopcho
Lisa M
LM
Yang
Tao
T
Copeland
Robert A
RA
Glass
Brian M
BM
Combs
Andrew P
AP
Falahatpisheh
Nikoo
N
Thompson
Lorin
L
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors*; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D010434:Pepsin A / Q000037:antagonists & inhibitors*; D010436:Pepstatins / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010880:Piperidines / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013552:Swine; D013997:Time Factors
D004791:Enzyme Inhibitors; D010436:Pepstatins; D010455:Peptides; D010880:Piperidines; D016282:Aspartic Acid Endopeptidases; D010434:Pepsin A; C031375:pepstatin