ABSTRACT
The proteinase from HIV undergoes rapid and irreversible deactivation caused by mild mechanical stirring. Both the free enzyme and the ternary Michaelis complex disappear in two separate first-order processes, with half-times of 3.0 and 0.8 minutes, respectively. Ignoring these deactivation steps distorts the results of kinetic analyses.
Subject(s)
HIV Protease Inhibitors/pharmacology , HIV Protease/metabolism , Amino Acid Sequence , Catalysis , Hydrolysis , Kinetics , Molecular Sequence Data , Substrate SpecificityABSTRACT
We have discovered that 17beta-[N,N-(diethyl)carbamoyl]-6-azaandrost-4-en-3-one is a time-dependent inhibitor of type II 5alpha-reductase, as is the drug finasteride. Unlike finasteride, the 6-aza-steroid is not a time-dependent inhibitor of type I 5 alpha-reductase. Finasteride inhibition of type II enzyme proceeds in a two-step mechanism. At pH 6 and 37 degrees C, an initial finasteride-reductase complex is formed with a K(i)(app) of 11.9 +/- 4.1 nM. In a second step, an irreversible complex is formed with a rate constant of inactivation of 0.09 +/- 0.01 s(-1). In contrast, the 6-aza-steroid is a reversible inhibitor. From the results of a simplified mathematical analysis, based on the rapid equilibrium approximation, the inhibitor and the enzyme form an initial complex with a K(i) of 6.8 +/- 0.2 nM. The reversible formation of a final complex, with an overall K(i) of 0.07 +/- 0.02 nM, is characterized by a first-order isomerization rate constant 0.0035 +/- 0.0001 s(-1) for the forward step and 0.00025 +/- 0.00006 s(-1) for the backward step. All rate constants for the two-step mechanism were obtained by using a general numerical integration method. The best fit values for the association and dissociation rate constants were 5.0 microM(-1) s(-1) and 0.033 +/- 0.008 s(-1), respectively, and the isomerization rate constants were 0.0035 +/- 0.007 s(-1) and 0.000076 +/- 0.000019 s(-1). These values correspond to an initial K(i) of 6.5 nM and an overall dissociation constant of 0.14 nM. The data presented here show that both finasteride and the 6-aza-steroid analogs are potent against type II 5alpha-reductase, although their mechanisms of inhibition are different.
Subject(s)
5-alpha Reductase Inhibitors , Azasteroids/chemistry , Enzyme Inhibitors/chemistry , Finasteride/chemistry , Humans , Isoenzymes/antagonists & inhibitors , Kinetics , Structure-Activity RelationshipABSTRACT
The intrinsic fluorescence of tyrosine increases by a factor of approximately two when the carboxy group is liberated from a peptide bond by hydrolysis. The increase in fluorescence provides a novel way to monitor the hydrolysis of native tyrosine peptides that contain only proteinogenic amino acids. Thus, for example, the hydrolysis by HIV-1 proteinase of a heptapeptide viral protein fragment gag129-135, Ser-Gln-Asn-Tyr-Pro-Ile-Val, was followed continuously at excitation and emission wavelengths 275 and 305 nm. The fluorescence increase is magnified by at least a factor of a thousand when a resonance energy quencher, such as paranitrophenylalanine, is in the vicinity. For example, the peptide Lys-Ala-Arg-Val-Tyr-Phe(p-NO2)-Glu-Ala-Nle-NH2 [Richards et al. (1990) J. Biol. Chem. 265, 7733], widely used for spectrophotometric assays of the HIV-1 proteinase, yields a substrate:product fluorescence ratio greater than 1:1000. Tyrosine-containing substrates of pepsin and trypsin showed similar behavior. The detection limit of the present method is at least one order of magnitude lower than absorbance assays of p-nitrophenylalanine peptides.