Rate-determining steps in HIV-1 protease catalysis. The hydrolysis of the most specific substrate

Research output: Article

36 Citations (Scopus)

Abstract

The human immunodeficiency virus type-1 (HIV-1) encodes a protease which is essential for the production of infectious virus. The protease prefers substrates that contain glutamic acid or glutamine at the P2' position. The catalytic role of these residues has been studied by using a highly specific fluorogen substrate, 2-aminobenzoyl-Thr-Ile-Nle-Phe(NO2)-Gln-Arg (substrate QR), and its counterpart (substrate ER) containing Glu in place of Gln. The newly designed substrate ER that contains a pair of charged residues at P2' and P3' sites is the most specific substrate described so far for HIV-1 protease. The specificity rate constant (k(cat)/K(m) = 2.1 x 107 M-1 s- 1) approaches, but does not reach, the diffusion limit. This follows from the appreciable solvent kinetic deuterium isotope effects on the rate constants, indicating 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 concentration from a physical to chemical step, while the negative activation entropy becomes positive. The rate increase with substrate ER is 50-fold with respect to substrate QR in the presence of 0.1 M NaCl and diminishes to 3.5- fold at 2.0 M NaCl concentration, as a consequence of a considerable rate increase at high salt concentration with substrate QR but not with substrate ER. The K(m) value is much lower for the substrate ER (0.8 μM) than for substrate QR (15 μM), indicating a more effective binding for substrate ER at 0.1 M NaCl. Unexpectedly, the 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 acid may be utilized in designing inhibitors for therapeutic purposes.

Original languageEnglish
Pages (from-to)32180-32184
Number of pages5
JournalJournal of Biological Chemistry
Volume271
Issue number50
DOIs
Publication statusPublished - 1996

Fingerprint

Catalysis
Viruses
HIV-1
Hydrolysis
Peptide Hydrolases
Salts
Entropy
Glutamic Acid
Substrates
Chemical Phenomena
Deuterium
Glutamine
Isotopes
Rate constants
Chemical activation
Therapeutics

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{bcf1bc53cd754103a477b86d97e5d86b,
title = "Rate-determining steps in HIV-1 protease catalysis. The hydrolysis of the most specific substrate",
abstract = "The human immunodeficiency virus type-1 (HIV-1) encodes a protease which is essential for the production of infectious virus. The protease prefers substrates that contain glutamic acid or glutamine at the P2' position. The catalytic role of these residues has been studied by using a highly specific fluorogen substrate, 2-aminobenzoyl-Thr-Ile-Nle-Phe(NO2)-Gln-Arg (substrate QR), and its counterpart (substrate ER) containing Glu in place of Gln. The newly designed substrate ER that contains a pair of charged residues at P2' and P3' sites is the most specific substrate described so far for HIV-1 protease. The specificity rate constant (k(cat)/K(m) = 2.1 x 107 M-1 s- 1) approaches, but does not reach, the diffusion limit. This follows from the appreciable solvent kinetic deuterium isotope effects on the rate constants, indicating 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 concentration from a physical to chemical step, while the negative activation entropy becomes positive. The rate increase with substrate ER is 50-fold with respect to substrate QR in the presence of 0.1 M NaCl and diminishes to 3.5- fold at 2.0 M NaCl concentration, as a consequence of a considerable rate increase at high salt concentration with substrate QR but not with substrate ER. The K(m) value is much lower for the substrate ER (0.8 μM) than for substrate QR (15 μM), indicating a more effective binding for substrate ER at 0.1 M NaCl. Unexpectedly, the 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 acid may be utilized in designing inhibitors for therapeutic purposes.",
author = "Z. Szeltner and L. Polg{\'a}r",
year = "1996",
doi = "10.1074/jbc.271.50.32180",
language = "English",
volume = "271",
pages = "32180--32184",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "50",

}

TY - JOUR

T1 - Rate-determining steps in HIV-1 protease catalysis. The hydrolysis of the most specific substrate

AU - Szeltner, Z.

AU - Polgár, L.

PY - 1996

Y1 - 1996

N2 - The human immunodeficiency virus type-1 (HIV-1) encodes a protease which is essential for the production of infectious virus. The protease prefers substrates that contain glutamic acid or glutamine at the P2' position. The catalytic role of these residues has been studied by using a highly specific fluorogen substrate, 2-aminobenzoyl-Thr-Ile-Nle-Phe(NO2)-Gln-Arg (substrate QR), and its counterpart (substrate ER) containing Glu in place of Gln. The newly designed substrate ER that contains a pair of charged residues at P2' and P3' sites is the most specific substrate described so far for HIV-1 protease. The specificity rate constant (k(cat)/K(m) = 2.1 x 107 M-1 s- 1) approaches, but does not reach, the diffusion limit. This follows from the appreciable solvent kinetic deuterium isotope effects on the rate constants, indicating 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 concentration from a physical to chemical step, while the negative activation entropy becomes positive. The rate increase with substrate ER is 50-fold with respect to substrate QR in the presence of 0.1 M NaCl and diminishes to 3.5- fold at 2.0 M NaCl concentration, as a consequence of a considerable rate increase at high salt concentration with substrate QR but not with substrate ER. The K(m) value is much lower for the substrate ER (0.8 μM) than for substrate QR (15 μM), indicating a more effective binding for substrate ER at 0.1 M NaCl. Unexpectedly, the 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 acid may be utilized in designing inhibitors for therapeutic purposes.

AB - The human immunodeficiency virus type-1 (HIV-1) encodes a protease which is essential for the production of infectious virus. The protease prefers substrates that contain glutamic acid or glutamine at the P2' position. The catalytic role of these residues has been studied by using a highly specific fluorogen substrate, 2-aminobenzoyl-Thr-Ile-Nle-Phe(NO2)-Gln-Arg (substrate QR), and its counterpart (substrate ER) containing Glu in place of Gln. The newly designed substrate ER that contains a pair of charged residues at P2' and P3' sites is the most specific substrate described so far for HIV-1 protease. The specificity rate constant (k(cat)/K(m) = 2.1 x 107 M-1 s- 1) approaches, but does not reach, the diffusion limit. This follows from the appreciable solvent kinetic deuterium isotope effects on the rate constants, indicating 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 concentration from a physical to chemical step, while the negative activation entropy becomes positive. The rate increase with substrate ER is 50-fold with respect to substrate QR in the presence of 0.1 M NaCl and diminishes to 3.5- fold at 2.0 M NaCl concentration, as a consequence of a considerable rate increase at high salt concentration with substrate QR but not with substrate ER. The K(m) value is much lower for the substrate ER (0.8 μM) than for substrate QR (15 μM), indicating a more effective binding for substrate ER at 0.1 M NaCl. Unexpectedly, the 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 acid may be utilized in designing inhibitors for therapeutic purposes.

UR - http://www.scopus.com/inward/record.url?scp=0029753776&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029753776&partnerID=8YFLogxK

U2 - 10.1074/jbc.271.50.32180

DO - 10.1074/jbc.271.50.32180

M3 - Article

C2 - 8943273

AN - SCOPUS:0029753776

VL - 271

SP - 32180

EP - 32184

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 50

ER -