David J. T. Porter

Changes in human immunodeficiency virus type 1 Gag at positions L449 and P453 are linked to I50V protease mutants in vivo and cause reduction of sensitivity to amprenavir and improved viral fitness in vitro.

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Gag protease cleavage sites (CS) undergo sequence changes during the development of resistance to several protease inhibitors (PIs). We have analyzed the association of sequence variation at the p7/p1 and p1/p6 CS in conjunction with amprenavir (APV)-specific protease mutations following PI combination therapy with APV. Querying a central resistance data repository resulted in the detection of significant associations (P < 0.001) between the presence of APV protease signature mutations and Gag L449F (p1/p6 LP1′F) and P453L (p1/p6 PP5′L) CS changes. In population-based sequence analyses the I50V mutant was invariably linked to either L449F or P453L. Clonal analysis revealed that both CS mutations were never present in the same genome. Sequential plasma samples from one patient revealed a transition from I50V M46L P453L viruses at early time points to I50V M46I L449F viruses in later samples. Various combinations of the protease and Gag mutations were introduced into the HXB2 laboratory strain of HIV-1. In both single- and multiple-cycle assay systems and in the context of I50V, the L449F and P453L changes consistently increased the 50% inhibitory concentration of APV, while the CS changes alone had no measurable effect on inhibitor sensitivity. The decreased in vitro fitness of the I50V mutant was only partially improved by addition of either CS change (I50V M46I L449F mutant replicative capacity ≈ 16% of that of wild-type virus). Western blot analysis of Pr55 Gag precursor cleavage products from infected-cell cultures indicated accumulation of uncleaved Gag p1-p6 in all I50V viruses without coexisting CS changes. Purified I50V protease catalyzed cleavage of decapeptides incorporating the L449F or P453L change 10-fold and 22-fold more efficiently than cleavage of the wild-type substrate, respectively. HIV-1 protease CS changes are selected during PI therapy and can have effects on both viral fitness and phenotypic resistance to PIs.

7 Flavoprotein Oxidases

Publisher Summary The concept of a flavoprotein oxidase is easier to understand than to define by international rules. A flavoprotein or flavoenzyme is commonly understood to mean an apoenzyme which together with its more or less tightly attached flavin coenzyme, catalyzes a redox reaction during which either one or two electrons from the electron donor are transferred transiently to the isoalloxazine nucleus of the flavin coenzyme and then to the electron acceptor. All flavoproteins belong to the class oxidoreductases. The term “oxidase” is a recommended name for an oxidoreductase which utilizes O 2 as the electron acceptor. A flavoprotein oxidase is defined as a flavoprotein. The definition singles out the simple flavoprotein oxidases in which flavin are the only recognizable prosthetic group that transiently accepts electrons originating in the donor substrate. The emphasis of this chapter concerns the kinetic and chemical mechanism of flavoprotein oxidase catalysis. There are several cogent reasons for such relatively restricted coverage. In the case of flavin, recent advances in the understanding of the mechanism of both the enzymic and nonenzymic reactions have resulted in a most productive liaison between the two approaches. In addition, this chapter discusses enzymological and model studies related to the mechanism of action of only three flavoprotein oxidases—namely, glucose oxidase, L-amino acid oxidase, and D-amino acid oxidase.

Nitro analogs of substrates for adenylosuccinate synthetase and adenylosuccinate lyase

The reactivities of the nitro analogs of the substrates of adenylosuccinate synthetase and adenylosuccinate lyase, the enzymes which catalyze the penultimate and last step, respectively, in the pathway for AMP biosynthesis have been examined. Alanine-3-nitronate, an aspartate analog, was a substrate for the synthetase from Azotobacter vinelandii, having a kcat/Km which was approximately 30% that for aspartate. The product of this reaction was N6-(L-1-carboxy-2-nitroethyl)-AMP. Of nine other substrate analogs tested, only cysteine sulfinate (having 5.5% of the activity of aspartate) was reactive. These results demonstrate the strict requirement of the synthetase for a negatively charged substituent, with a carboxylate-like geometry, at the beta-carbon of the alpha-amino acid substrate. The lyase, purified to homogeneity from brewers yeast by a new procedure, did not utilize N6-(L-1-carboxy-2-nitroethyl)-AMP as a substrate. However, the nitronate form of this analog was a good inhibitor of the lyase (Km/Ki = 28 when compared to adenylosuccinate), suggesting that it mimics a carbanionic intermediate in the reaction pathway. The avid binding of bromphenol blue by the lyase (Ki = 0.95 microM) was used for active site titrations and for displacement of the enzyme, in the purification protocol, from blue Sepharose.

The bioorganic chemistry of the nitroalkyl group

Abstract Nitroalkyl groups are conspicuously rare among pharmaceutical agents, and the bioactivity of substituted nitroalkanes has been described in few instances. This article examines the natural occurrence of such compounds and the reactions of nitroalkyl compounds with enzymes. The discussion is not limited to nitro hydrocarbons; the term “nitroalkyl” is intended to distinguish the compounds from nitroaromatic compounds, which are better known in pharmacology and involve different chemical considerations. Further study of the bioorganic chemistry of the nitroalkyl group may contribute to the understanding of biosynthetic strategies and enzymatic catalysis and may permit the rational design of useful bioactive molecules.

Location of hydrogen transfer steps in the mechanism of reduction of L-amino acid oxidase.

Abstract We show by means of a specific kinetic isotope effect that the bond between hydrogen and the 2-carbon of the substrate is broken in forming the 540 mμ complex of L-amino acid oxidase. In addition, this process is considerably slowed in 2 H 2 O. The conversion of the 540 mμ complex to the fully reduced enzyme appears not to involve the rate-limiting transfer of a species of hydrogen of any kind.

Inactivation of alanine aminotransferase by the neurotoxin β-cyano-L-alanine

Abstract β-Cyano-L-alanine inactivates pig heart alanine aminotransferase. The nitrile and enzyme form a freely dissociable Michaelis complex which rearranges to a form of inactive enzyme. The inactivated enzyme slowly recovers activity at 25° in 100 mM phosphate buffer, pH 7.4. The observations are consistent with a mechanism of inactivation similar to that thought to apply to the suicide inactivator propargylglycine except that the putative covalent modification of the apoenzyme is relatively labile in the case of the nitrile.

Inhibition of succinate dehydrogenase by nitroacetate and by the toxic antibiotic nitraminoacetate

Summary Nitraminoacetate and the nitronate of nitroacetate are effective inhibitors of beef heart succinate dehydrogenase, respectively binding about 17 and 6.6 times more tightly than succinate at pH 8.0 and 25° as judged by the K m /K i ratios. Unlike the case of the suicide inactivator 3-nitropropionate, these compounds are freely reversible inhibitors. The toxicity of nitraminoacetate, an antibiotic elaborated by Streptomyces noursei , may be secondary to the inhibition of the Krebs cycle at the succinate dehydrogenase reaction, and the nitramino group may prove useful as a carboxylate analog for other enzymes.

The kinetics of imino acid accumulation in the D-amino acid oxidase reaction*

When the oxidation of D-phenylalanine by D-amino acid oxidase is measured in stopped-flow turnover experiments there is a lag in the formation of keto-phenylpyruvate which becomes progressively greater as the pH is raised from 6.5. Borohydride trapping experiments show that the transient accumulation of free imino acid largely accounts for the lag at pH 8.7. These results are consistent with the known hydrolytic behavior of imines.

Reduction of D-amino acid oxidase by β-chloroalanine: Enhancement of the reduction rate by cyanide

Abstract Using rapid reaction techniques, it is shown that there is no effect of cyanide on the first three phases of the anaerobic reaction of β-chloroalanine with the enzyme. However, the rate of appearance of free fully reduced enzyme, following the third phase, is increased 50-fold by cyanide and shows saturation kinetics with respect to cyanide. To account for this increased reductive rate in the presence of cyanide, it is proposed that cyanide attacks an enzyme-bound dehydroalanine formed in the elimination reaction.

Oxidation of N-nitroethylenediamine, a GABA analog from Agaricussilvaticus, by GABA aminotransferase

Abstract N-Nitroethylenediamine is a mushroom product which closely resembles the neurotransmitter 4-aminobutyrate, GABA. The nitramine is sequentially accepted as a substrate by the GABA-catabolizing enzymes GABA aminotransferase (EC 2.6.1.19) and succinic semialdehyde dehydrogenase (EC 1.2.1.16). In view of the steric and ionic similarity of the nitramino group to the carboxymethyl group, nitramines may prove generally useful for enzymological and pharmacological purposes as analogs of carboxylic acids.