Constance D. Anderson

Selective inactivation of rabbit muscle glycerophosphate dehydrogenase by diazotized 3-aminopyridine adenine dinucleotide☆

Abstract Rabbit muscle glycerophosphate dehydrogenase was rapidly and irreversibly inactivated at pH 7.0 and 4 °C by low concentrations of diazotized 3-aminopyridine adenine dinucleotide. The enzyme was protected from inactivation by the presence of NAD. During the inactivation process, 2 mol of diazotized 3-aminopyridine adenine dinucleotide was covalently attached per 1 mol of enzyme or one diazotized 3-aminopyridine adenine dinucleotide per active site. The selective modification of an active-site cysteine residue was indicated by the observation that one sulfhydryl group per active site was lost during the inactivation process. Diazotized 3-aminopyridine adenine dinucleotide did not irreversibly inactivate bovine lactate dehydrogenase, M 4 isozyme, or bovine heart mitochondrial malate dehydrogenase, presumably due to the inaccessibility of active-site sulfhydryl groups. Diazotized 3-aminopyridine adenine dinucleotide was selectively bound to the lactate dehydrogenase as a coenzyme-competitive inhibitor.

Azotobacter vinelandii glucose 6-phosphate dehydrogenase properties of NAD- and NADP-linked reactions

Glucose 6-phosphate oxidation, catalyzed by purified Azotobacter vinelandii glucose 6-phosphate dehydrogenase, was studied with respect to the selective utilization of NAD, NADP, thionicotinamide adenine dinucleotide or thionicotinamide adenine dinucleotide phosphate as coenzyme. A sigmoidal relationship was observed for the effect of substrate concentration on initial velocities when either NAD, NADP or thionicotinamide adenine dinucleotide was used as coenzyme, with N values from the Hill equation equalling 2.0, 1.7, and 1.7, respectively. The thionicotinamide analogs of NAD and NADP both functioned as coenzyme-competitive inhibitors of the enzyme-catalyzed NAD- and NADP-linked reactions. A dual wavelength assay, using a combination of NADP and thio-NAD, was established and was used to demonstrate that increasing glucose 6-phosphate concentration did not change the enzyme preference for the coenzyme form used. Sigmoidal relationships were observed for reduction of both dinucleotides, and N values were the same as those observed when each dinucleotide was studied as the only coenzyme form present in reaction mixtures. Using the dual wavelength assay, inhibition by isocitrate, 6-phosphogluconate, ATP, and palmitoyl-CoA was shown to be equally effective in both NAD- and NADP-linked reactions. An enzyme activator, glucosamine 6-phosphate, altered the glucose 6-phosphate sigmoidicity through activation at low substrate concentrations.

Kinetic studies of Haemophilus influenzae 6-phosphogluconate dehydrogenase

Haemophilus influenzae 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP+ 2-oxidoreductase (decarboxylating), EC 1.1.1.44) was purified 308-fold to electrophoretic homogeneity with a 16% recovery through a five-step procedure involving salt fractionation and hydrophobic and affinity chromatography. The purified enzyme was demonstrated to be a dimer of Mr 70,000, and to catalyze a sequential reaction process. The enzyme was NADP-specific and kinetic parameters for the oxidation of 6-phosphogluconate were determined for NADP and four structural analogs of NADP. Coenzyme-competitive inhibition by adenosine derivatives was significantly enhanced by the presence of a 2-phosphoryl group consistent with the observed coenzyme specificity of the enzyme. The purified enzyme was effectively inhibited by 3-aminopyridine adenine dinucleotide phosphate, but at concentrations higher than that observed to inhibit growth of the organism. Rates of inactivation of the enzyme by N-ethylmaleimide were suggestive of sulfhydryl involvement in the reaction catalyzed.

Nonpolar interactions in the modification of an essential sulfhydryl of sorbitol dehydrogenase by N-alkylmaleimides

A series of N-alkylmaleimides varying in chainlength from N-methyl- to N-octylmaleimide inclusive was shown to effectively inactivate sheep liver sorbitol dehydrogenase at pH 7.5 and 25 degrees C. The apparent second-order rate constants for inactivation increased with increasing chainlength of the N-alkylmaleimide used. Positive chainlength effects were also indicated by the Kd values for the N-ethyl and N-heptyl derivatives obtained from studies of the saturation kinetics observed for inactivation of the enzyme at high concentrations of these maleimides. The complete inactivation of sorbitol dehydrogenase was demonstrated to occur through the selective covalent modification of one cysteine residue per subunit of enzyme. The stoichiometry of enzyme inactivation was supported on the one hand by fluorescence titration with fluorescein mercuric acetate of the native and the inactivated enzyme, and, on the other hand, by the simultaneous inactivation of the enzyme with selective modification of one sulfhydryl per subunit by N-[p-(2-benzoxazolyl)phenyl]maleimide. Protection of the enzyme from N-alkylmaleimide inactivation was observed with the binding of NADH, whereas both NAD and sorbitol were ineffective as protecting ligands. Diazotized 3-aminopyridine adenine dinucleotide, in contrast to previous studies of this reagent with yeast alcohol dehydrogenase and rabbit muscle glycerophosphate dehydrogenase, did not function as a site-labeling reagent for sorbitol dehydrogenase.

Acridine dyes as effectors in papain catalysis

Abstract The catalytic activity of the sulfhydryl protease, papain, toward the synthetic ester substrate, α- N -benzoyl- l -arginine ethyl ester, is enhanced by several acridine dyes. Kinetic studies on the effects of proflavine (3,6-diaminoacridine) on the papain-catalyzed hydrolysis at pH 6.6 (25 °C) of both α- N -benzoyl- l -arginine ethyl ester and N -benzoylglycine ethyl ester indicate that the dye primarily affects K m (app) , having little or no effect upon k ca t . On the other hand, proflavine appears to have no measurable effect upon the catalytic activity of papain toward N -benzyloxycarbonylglycine p -nitrophenyl ester. Quinacrine hydrochloride appears to behave much like proflavine in theenhancement of papain-catalyzedbenzoylarginine ethyl ester hydrolysis. These findings are interpreted in terms of possible effects of dye on individual rate constants in the “acyl enzyme” scheme which characterizes papain action, and they are compared with reported findings concerning the rate-enhancing effects of proflavine on the related sulfhydryl protease ficin.

Sulfhydryl enzyme inactivation by nicotinoylacrylates

Abstract Nicotinoylacrylic acid and the corresponding methyl, ethyl, propyl and benzyl esters were studied with respect to selective inactivation of dehydrogenases through covalent modification of essential sulfhydryl groups. An effective inactivation of yeast alcohol dehydrogenase, yeast glutathione reductase and yeast 6-phosphogluconate dehydrogenase was observed with methyl and ethyl nicotinoylacrylates, with nicotinoylacrylic acid and the larger propyl and benzyl esters being considerably less effective. Fluorescein mercuric acetate titration studies of inactivated yeast alcohol dehydrogenase and studies of the oxidized (disulfide) form of yeast glutathione reductase were consistent with inactivation processes involving sulfhydryl modification. Protection against nicotinoylacrylate inactivation was afforded by the binding of ligands to either the coenzyme or substrate binding sites. Inactivation of yeast alcohol dehydrogenase by ethyl nicotinoylacrylate exhibited an unexpected greater selectivity, resulting in the covalent modification of only one of the two reactive sulfhydryl groups at the catalytic site.

Properties and applications of immobilized snake venom NAD glycohydrolase

The NAD glycohydrolase (NADase) from Bungarus fasciatus snake venom was adsorbed on concanavalin A-Sepharose, and demonstrated to retain both hydrolase and transglycosidase activities in the bound form. The matrix-bound enzyme was stable to repeated washing with buffer and storage at 4 degrees C. The bound enzyme exhibited the same Km value for hydrolysis of nicotinamide-1,N6-ethenoadenine dinucleotide as previously measured with the soluble, purified form of the enzyme. The bound NADase was used repeatedly for a preparative-scale synthesis of 3-acetylpyridine adenine dinucleotide. It was further demonstrated that the immobilized enzyme could be prepared directly from crude snake venom, thus avoiding the time required for purification. The application of the immobilized snake venom NADase for the preparation of pyridine nucleotide coenzyme analogs has many advantages over procedures used previously for analog synthesis.

Purification of ficin by affinity chromatography

Abstract The sulfhydryl proteinase ficin (EC 3.4.4.12) was purified by chromatography on an agarose-mercurial column. Two separate protein fractions were eluted, ficin and mercurificin, both exhibiting enzymatic activity upon activation by excess thiol.

3-aminopyridine 1,N6-ethenoadenine dinucleotide phosphate: a fluorescent reagent for NADP-requiring enzymes.

3-Aminopyridine 1,N6-ethenoadenine dinucleotide phosphate (epsilon-AADP) was synthesized from 3-aminopyridine adenine dinucleotide phosphate by reaction with chloroacetaldehyde. The reaction was monitored by high-pressure liquid chromatography. The product, epsilon-AADP, was purified by ion-exchange chromatography followed by a Sephadex G-10 desalting process. Spectrophotometric and fluorimetric properties of epsilon-AADP were obtained at neutral pH. Diazotization of epsilon-AADP was documented by azodye formation at pH values below 3 and the diazotized derivative was shown to react rapidly with sulfhydryl compounds at neutrality. As an analog of NADP, epsilon-AADP was demonstrated to be an effective NADP-competitive inhibitor of NADP-requiring dehydrogenases, reductases, and an NAD glycohydrolase.

Preparation and characterization of the NAD vinylogue, 3-pyridylacryloamide adenine dinucleotide☆

The vinylogue of NAD, 3-pyridylacryloamide adenine dinucleotide, was prepared from NAD and 3-pyridylacryloamide through the snake venom NADase-catalyzed transglycosidation reaction. The analog, purified by ion-exchange chromatography, was obtained in a 55% yield. The cyanide adduct and reduced form of the analog exhibited absorbance maxima at 358 nm and 378 nm, respectively, with extinction coefficients in each case being 2.3-times higher than those reported for the corresponding NAD derivatives. 3-Pyridylacryloamide adenine dinucleotide served as a coenzyme with bovine liver glutamic dehydrogenase and to a lesser extent with malate and lactate dehydrogenases. The analog was not reduced in reactions catalyzed by yeast and horse liver alcohol dehydrogenases, sheep liver sorbitol dehydrogenase, and rabbit muscle glycerophosphate dehydrogenase. Substitution of the pyridylacryloamide analogs for NAD and NADH in the assay of substrates for glutamic dehydrogenase was demonstrated.