Human immunodeficiency virus-1 protease. 1. Initial velocity studies and kinetic characterization of reaction intermediates by 18O isotope exchange.
The peptidolytic reaction of HIV-1 protease has been investigated by using four oligopeptide substrates, Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, Ac-Ser-Gln-Ser-Tyr-Pro-Val-Val-NH2, and Ac-Arg-Lys-Ile-Leu-Phe-Leu-Asp-Gly-NH2, that resemble two cleavage sites found within the naturally occurring polyprotein substrates Pr55gag and Pr160gag-pol. The values for the kinetic parameters V/KEt and V/Et were 0.16-7.5 mM-1 s-1 and 0.24-29 s-1, respectively, at pH 6.0, 0.2 M NaCl, and 37 degrees C. By use of a variety of inorganic salts, it was concluded that the peptidolytic reaction is nonspecifically activated by increasing ionic strength. V/K increased in an apparently parabolic fashion with increasing ionic strength, while V was either increased or decreased slightly. From product inhibition studies, the kinetic mechanism of the protease is either random or ordered uni-bi, depending on the substrate studied. The reverse reaction or a partial reverse reaction (as measured by isotope exchange of the carboxylic product into substrate) was negligible for most of the oligopeptide substrates, but the enzyme catalyzed the formation of Ac-Ser-Gln-Asn-Tyr-Phe-Leu-Asp-Gly-NH2 from the products Ac-Ser-Gln-Asn-Tyr and Phe-Leu-Asp-Gly-NH2. The protease-catalyzed exchange of an atom of 18O from H2 18O into the re-formed substrates occurred at a rate which was 0.01-0.12 times that of the forward peptidolytic reaction. The results of these studies are in accord with the formation of a kinetically competent enzyme-bound amide hydrate intermediate, the collapse of which is the rate-limiting chemical step in the reaction pathway.
Hyland
L J
LJ
Department of Medicinal Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.
Tomaszek
T A
TA
Roberts
G D
GD
Carr
S A
SA
Magaard
V W
VW
Bryan
H L
HL
Fakhoury
S A
SA
Moore
M L
ML
Minnich
M D
MD
Culp
J S
JS
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D004789:Enzyme Activation; D016333:HIV Protease / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D010103:Oxygen Isotopes
D009842:Oligopeptides; D010103:Oxygen Isotopes; D016333:HIV Protease