title: Two-step binding mechanism for HIV protease inhibitors.
abstract: Rate constants for binding of five inhibitors of human immunodeficiency virus (HIV) protease were determined by stopped-flow spectrofluorometry. The two isomers of quinoline-2-carbonyl-Asn-Phe psi-[CH(OH)CH2N]Pro-O-t-Bu (R diastereomer = 1R; S diastereomer = 1S) quenched the protein fluorescence of HIV protease and thus provided a spectrofluorometric method to determine their binding rate constants. The dissociation rate constants for acetyl-Thr-Ile-Leu psi(CH2NH)Leu-Gln-Arg-NH2 (2), (carbobenzyloxy)-Phe psi[CH(OH)CH2N]Pro-O-t-Bu (3), and pepstatin were determined by trapping free enzyme with 1R as 2, 3, and pepstatin dissociated from the respective enzyme.inhibitor complex. Association rate constants of 1R, 2, and pepstatin were calculated from the time-dependent inhibition of protease-catalyzed hydrolysis of the fluorescent substrate (2-aminobenzoyl)-Thr-Ile-Nle-Phe(NO2)-Gln-Arg-NH2 (4). The kinetic data for binding of 1S to the protease fit a two-step mechanism. Kd values for these inhibitors were calculated from the rate constants for binding and were similar to the respective steady-state Ki values.
authors.affiliation: Division of Experimental Therapy, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
authors.forename: E S
authors.initials: ES
authors.lastname: Furfine
authors.forename: E
authors.initials: E
authors.lastname: D'Souza
authors.forename: K J
authors.initials: KJ
authors.lastname: Ingold
authors.forename: J J
authors.initials: JJ
authors.lastname: Leban
authors.forename: T
authors.initials: T
authors.lastname: Spector
authors.forename: D J
authors.initials: DJ
authors.lastname: Porter
mesh_terms: D000595:Amino Acid Sequence; D016333:HIV Protease / Q000378:metabolism; D017320:HIV Protease Inhibitors; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D010436:Pepstatins / Q000378:metabolism; D011480:Protease Inhibitors / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013237:Stereoisomerism
chemical_list: D017320:HIV Protease Inhibitors; D009842:Oligopeptides; D010436:Pepstatins; D011480:Protease Inhibitors; C014195:Streptomyces pepsin inhibitor; D016333:HIV Protease; C031375:pepstatin