Use of nitrogen-15 kinetic isotope effects to elucidate details of the chemical mechanism of human immunodeficiency virus 1 protease

We have used 15N kinetic isotope effects of the HIV-1 protease-catalyzed peptidolysis of Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 to characterize the chemical mechanism of this enzyme. In addition, the multiple isotope effects have been determined by measuring the 15N kinetic isotope effects in both H2O and D2O. The isotope effects, measured on values of V/K, were determined by the incorporation of a radiolabel (tritium and 14C in peptides bearing the heavy and light isotopes, respectively) at a position remote from the isotopically labeled scissile peptide bond, such that the isotope effect was determined by measurement of the change in the 14C/3H ratio in recovered substrates at various fractions of reaction. At pH = 6.0 (37 degrees C), the nitrogen isotope effects were slightly, but significantly, inverse in both solvents: 15(V/K)H2O = 0.995 +/- 0.002, and 15(V/K)D2O = 0.992 +/- 0.003. The observation of an inverse nitrogen kinetic isotope effect implies that bonding to the nitrogen atom is becoming stiffened in a reaction transition state, and since this inverse isotope effect is enhanced in D2O, this isotope effect likely arises from protonation of the proline nitrogen atom.(ABSTRACT TRUNCATED AT 250 WORDS)