title: Synthesis and characterization of a selenium-containing substrate of alpha-chymotrypsin. Selenium-77 nuclear magnetic resonance observation of an acyl-alpha-chymotrypsin intermediate.
abstract: The selenium-containing ester p-nitrophenyl (phenylselenyl)acetate, C6H5SeCH2C(O)-OC6H4-p-(NO2), has been synthesized, characterized as a substrate for alpha-chymotrypsin (k2/KM = 15.2 X 10(3) M-1 s-1, KMapp = 5.16 X 10(-6) M, pH 7.77, 33% CH3CN, 25 degrees C), and shown to be an active-site titrant for the enzyme. A synthesis of the selenium-77 enriched p-nitrophenyl (phenylselenyl)acetate in 53% yield from 94.4% elemental selenium-77, followed by its reaction with alpha-chymotrypsin (pH 5.0, 0-3 degrees C), permitted the observation of the (phenylselenyl)acetyl-alpha-chymotrypsin reaction intermediate by selenium-77 NMR spectroscopy. This acyl-enzyme species had a chemical shift of 275.1 ppm relative to dimethyl selenide. Accompanying this resonance was a lower intensity, pH-dependent resonance that is assigned to (phenylselenyl)acetate on the basis of a pH titration of the model compound. Deacylation in the presence of hydrazine sulfate produced a resonance at 332.3 ppm in addition to the 302.2 ppm resonance of (phenylselenyl)acetate at pH 7.85. Denaturation of the acyl-enzyme resulted in a shift of the 275.1 ppm resonance to 334.6 ppm at pH 4.90, in good agreement with the selenium-77 chemical shift of the model compound, methyl (phenylselenyl)acetate, in CDCl3 (333.3 ppm). The large shielding observed for the native acyl-enzyme in comparison to the denatured species can be attributed to a resonance-perturbed ester linkage and/or steric compression at a nonbonding orbital of the selenium nucleus.
mesh_terms: D000818:Animals; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism*; D007554:Isotopes; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009596:Nitrophenols / Q000138:chemical synthesis* / Q000378:metabolism; D016566:Organoselenium Compounds; D010179:Pancreas / Q000201:enzymology; D011485:Protein Binding; D011489:Protein Denaturation; D012643:Selenium / Q000138:chemical synthesis* / Q000378:metabolism
chemical_list: D007554:Isotopes; D009596:Nitrophenols; D016566:Organoselenium Compounds; C050292:4-nitrophenyl(phenylselenyl)acetate; D002918:Chymotrypsin; D012643:Selenium