RATE-DETERMINING STEPS IN HIV-1 PROTEASE CATALYSIS - THE HYDROLYSIS OF THE MOST SPECIFIC SUBSTRATE

The human immunodeficiency virus type-1 (HIV-1) encodes a protease whi ch is essential for the production of infectious virus, The protease p refers substrates that contain glutamic acid or glutamine at the P2' p osition. The catalytic role of these residues has been studied by usin g a highly specific fluorogen substrate, 2-aminobenzoyl-Thr-Ile-Nle-Ph e(NO2)-Gln-Arg (substrate QR), and its counterpart (substrate ER) cont aining Glu in place of Gln. The newly designed substrate ER that conta ins a pair of charged residues at P2' and P3' sites is the most specif ic substrate described so far for HIV-1 protease. The specificity rate constant (k(cat)/K-m = 2.1 x 10(7) M(-1) s(-1)) approaches, but does not reach, the diffusion limit, This follows from the appreciable solv ent kinetic deuterium isotope effects on the rate constants, indicatin g that, independent of the salt concentration, the rate-limiting step of the catalysis is a chemical process rather than a physical one, The reaction also has positive entropy of activation, On the other hand, the rate-limiting step for substrate QR changes with increasing salt c oncentration from a physical to chemical step, while the negative acti vation entropy becomes positive, The rate increase with substrate ER i s 50-fold with respect to substrate QR in the presence of 0.1 M NaCl a nd diminishes to 3.5-fold at 2.0 M NaCl concentration, as a consequenc e of a considerable rate increase at high salt concentration with subs trate QR but not with substrate ER. The K-m value is much lower for th e substrate ER (0.8 pH) than for substrate QR (15 mu M), indicating a more effective binding for substrate ER at 0.1 M NaCl. Unexpectedly, t he strong binding appears to be achieved by the unionized form of Glu in P2', as follows from the remarkably different pH-rate profiles for substrates QR and ER, The effective binding elicited by the glutamic a cid may be utilized in designing inhibitors for therapeutic purposes.