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

Journal Article · Tue Aug 27 00:00:00 EDT 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00098a023· OSTI ID:5821883

Hyland, L J; Tomaszek, Jr, T A; 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 ^[1]

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1. Smline Beecham Pharmaceuticals, King of Prussia, PA (United States)

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.

• OSTI ID: 5821883
• Journal Information: Biochemistry; (United States), Vol. 30:34; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
AIDS VIRUS
BIOLOGICAL PATHWAYS
OXYGEN 18
ISOTOPE EFFECTS
PEPTIDE HYDROLASES
BIOCHEMICAL REACTION KINETICS
POLYPEPTIDES
REACTION INTERMEDIATES
ENZYMES
EVEN-EVEN NUCLEI
HYDROLASES
ISOTOPES
KINETICS
LIGHT NUCLEI
MICROORGANISMS
NUCLEI
ORGANIC COMPOUNDS
OXYGEN ISOTOPES
PARASITES
PEPTIDES
PROTEINS
REACTION KINETICS
STABLE ISOTOPES
VIRUSES
550201* - Biochemistry- Tracer Techniques