Human immunodeficiency virus-1 protease. 1. Initial velocity studies and kinetic characterization of reaction intermediates by sup 18 O isotope exchange

Human immunodeficiency virus-1 protease. 1. Initial velocity studies and kinetic characterization of reaction intermediates by sup 18 O isotope exchange

The peptidolytic reaction HIV-1 protease has been investigated by using four oligopeptide substrates, Ac-Ser-Gln-Asn-Pro-Val-Val-NH{sub 2}, Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH{sub 2}, Ac-Ser-Gln-Ser-Tyr-Pro-Val-Val-NH{sub 2}, and Ac-Arg-Lys-Ile-Leu-Phe-Leu-Asp-Gly-NH{sub 2} that resemble two c...

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Bibliographic Details
Published in:Biochemistry (Easton) Vol. 30:34
Main Authors: Hyland, L.J., Tomaszek, T.A. Jr, Roberts, G.D., Carr, S.A., Magaard, V.W., Bryan, H.L., Fakhoury, S.A., Moore, M.L., Minnich, M.D., Culp, J.S., DesJarlais, R.L., Meek, T.D.
Format: Journal Article
Language:English
Published: United States 27-08-1991
Subjects:
550201 - Biochemistry- Tracer Techniques
AIDS VIRUS
BASIC BIOLOGICAL SCIENCES
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL PATHWAYS
ENZYMES
EVEN-EVEN NUCLEI
HYDROLASES
ISOTOPE EFFECTS
ISOTOPES
KINETICS
LIGHT NUCLEI
MICROORGANISMS
NUCLEI
ORGANIC COMPOUNDS
OXYGEN 18
OXYGEN ISOTOPES
PARASITES
PEPTIDE HYDROLASES
PEPTIDES
POLYPEPTIDES
PROTEINS
REACTION INTERMEDIATES
REACTION KINETICS
STABLE ISOTOPES
VIRUSES
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Summary:The peptidolytic reaction HIV-1 protease has been investigated by using four oligopeptide substrates, Ac-Ser-Gln-Asn-Pro-Val-Val-NH{sub 2}, Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH{sub 2}, Ac-Ser-Gln-Ser-Tyr-Pro-Val-Val-NH{sub 2}, and Ac-Arg-Lys-Ile-Leu-Phe-Leu-Asp-Gly-NH{sub 2} that resemble two cleavage sites found within the naturally occurring polyprotein substrates Pr55{sup gag} and Pr160{sup gag-pol}. 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 protease-catalyzed exchange of an atom of {sup 18}O from H{sub 2}{sup 18}O into the re-formed substrates occurred at a rate which was 0.01-0.12 times that 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.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00098a023