Harold J. Bright

Insolubilized coenzymes: the covalent coupling of enzymatically active NAD to glass surfaces.

Abstract NAD was attached to the surface of glass beads by a diazo coupling procedure. The insolubilized NAD was shown to function as a coenzyme in the yeast alcohol dehydrogenase reaction.

Kinetic patterns of protocollagen hydroxylase and further studies on the polypeptide substrate

Abstract The kinetics of the protocollagen hydroxylase reaction were examined by the technique of measuring the initial velocities as a function of the concentration of one reactant with a series of fixed concentrations of a second reactant, and with a constant concentration of the remaining reactants. From the data a rate equation was developed for the interaction of the enzyme with ascorbate, α-ketoglutarate, oxygen, and polypeptide substrate. The kinetic data were consistent with a scheme involving consecutive bimolecular additions of ascorbate and α-ketoglutarate followed by the random order addition of oxygen and polypeptide substrate, but other kinetic schemes were not excluded. Studies with a purified enzyme system indicated that α-ketoglutarate was not consumed in stoichiometric amounts in the reaction. Further studies on the polypeptide substrate supported earlier indications that the enzyme can hydroxylate polypeptides without triple helical structure. When proline-labeled [14C]protocollagen was boiled and quenched, the synthesis of [14C]-hydroxyproline was 74% of the control value. Also, the polypeptide (Gly- l -Ala- l -Pro)n which apparently does not have triple helical structure in solution was found to serve as a substrate for the synthesis of hydroxyproline by the enzyme. The Km value and maximum velocity for (Gly- l -Ala- l -Pro)n were slightly lower than for ( l -Pro-Gly- l -Pro)n under the same conditions.

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.

Inactivation of alcohol dehydrogenase by 3-butyn-1-ol☆

Abstract Horse liver and yeast alcohol dehydrogenases are rapidly inactivated during their catalysis of the oxidation of 3-butyn-1-ol. In the case of the horse liver enzyme, the inactivation is secondary to covalent modification of the apoenzyme by an electrophilic product that accumulates in the reaction solution and that can also react with water, glutathione, and other enzymes. The modified protein exhibits enhanced ultraviolet absorbance, which is not bleached upon dialysis of the denatured enzyme at pH 7.4 for 24 h. The inactivation by 3-butyn-1-ol is more rapid than that which is afforded by the related alcohols 2-propyn-1-ol and 2-propen-1-ol under identical conditions and no inactivation is seen upon incubation with 3-hydroxypropanoic nitrile plus nicotinamide-adenine dinucleotide.

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.