Bruce M. Anderson

Reactions of the Neurospora crassa nitrate reductase with NAD(P) analogs.

The assimilatory NADPH-nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.3) from Neurospora crassa is competitively inhibited by 3-aminopyridine adenine dinucleotide (AAD) and 3-aminopyridine adenine dinucleotide phosphate (AADP) which are structural analogs of NAD and NADP, respectively. The amino group of the pyridine ring of AAD(P) can react with nitrous acid to yield the diazonium derivative which may covalently bind at the NAD(P) site. As a result of covalent attachment, diazotized AAD(P) causes time-dependent irreversible inactivation of nitrate reductase. However, only the NADPH-dependent activities of the nitrate reductase, i.e. the overall NADPH-nitrate reductase and the NADPH-cytochrome c reductase activities, are inactivated. The reduced methyl viologen- and reduced FAD-nitrate reductase activities which do not utilize NADPH are not inhibited. This inactivation by diazotized AADP is prevented by 1 mM NADP. The inclusion of 1 muM FAD can also prevent inactivation, but the FAD effect differs from the NADP protection in that even after removal of the exogenous FAD by extensive dialysis or Sephadex G-25 filtration chromatography, the enzyme is still protected against inactivation. The FAD-generated protected form of nitrate reductase could again be inactivated if the enzyme was treated with NADPH, dialyzed to remove the NADPH, and then exposed to diazotized AADP. When NADP was substituted for NADPH in this experiment, the enzyme remained in the FAD-protected state. Difference spectra of the inactivated nitrate reductase demonstrated the presence of bound AADP, and titration of the sulfhydryl groups of the inactivated enzyme revealed that a loss of accessible sulfhydryls had occurred. The hypothesis generated by these experiments is that diazotized AADP binds at the NADPH site on nitrate reductase and reacts with a functional sulfhydryl at the site. FAD protects the enzyme against inactivation by modifying the sulfhydryl. Since NADPH reverses this protection, it appears the modifications occurring are oxidation-reduction reactions. On the basis of these results, the physiological electron flow in the nitrate reductase is postulated to be from NADPH via sulfhydryls to FAD and then the remainder of the electron carriers as follows: NADPH leads to -SH leads to FAD leads to cytochrome b-557 leads to Mo leads to NO-3.

Studies of 3-Aminopyridine adenine dinucleotide phosphate

Summary3-Aminopyridine adenine dinucleotide phosphate (AADP) was prepared from NADP and 3-aminopyridine through the pig brain NADase-catalyzed pyridine base exchange reaction. The purified dinucleotide was chemically characterized and spectral properties of the compound were determined. The importance of the application of AADP in studies of NADP-requiring biochemical processes was indicated by the demonstration of AADP as an effective inhibitor of five NADP-requiring enzymes, by the demonstration of the fluorescence enhancement on the binding of AADP to yeast glucose-6-phosphate dehydrogenase when glucose-6-phosphate is present, and by the functioning of AADP as a fluorimetric substrate for snake venom nucleotide pyrophosphatase.

Maleimideinactivation of lactate dehydrogenase isozymes

Abstract The homologous beef lactate dehydrogenase (EC 1.1.1.27) isozymes H 4 and M 4 were observed to be effectively inactivated by a homologous series of N- alkylmaleimides . With each isozyme, the second-order rate constants of inactivation increased with increasing chain length of the alkyl group of the maleimide derivative. The rate of inactivation of lactate dehydrogenase (H 4 ) by N- heptylmaleimide was decreased in the presence of NADH while no protective effect by NADH was noted in the inactivation of lactate dehydrogenase (M 4 ) by this maleimide. Adenosine, AMP, ADP and adenosine diphosphoribose were shown to be coenzyme-competitive inhibitors of both lactate dehydrogenase (H 4 ) and lactate dehydrogenase (M 4 ). Binding patterns of these compounds were very similar with the two isozymes studied.

Use of competitive dead-end inhibitors to determine the chemical mechanism of action of yeast alcohol dehydrogenase

In this work, we have postulated a comprehensive and unified chemical mechanism of action for yeast alcohol dehydrogenase (EC 1.1.1.1, constitutive, cytoplasmic), isolated from Saccharomyces cerevisiae. The chemical mechanism of yeast enzyme is based on the integrity of the proton relay system: His-51....NAD+....Thr-48....R.CH2OH(H2>O)....Zn++, stretching from His-51 on the surface of enzyme to the active site zinc atom in the substrate-binding site of enzyme. Further, it is based on extensive studies of steady-state kinetic properties of enzyme which were published recently. In this study, we have reported the pH-dependence of dissociation constants for several competitive dead-end inhibitors of yeast enzyme from their binary complexes with enzyme, or their ternary complexes with enzyme and NAD+ or NADH; inhibitors include: pyrazole, acetamide, sodium azide, 2-fluoroethanol, and 2,2,2-trifluorethanol. The unified mechanism describes the structures of four dissociation forms of apoenzyme, two forms of the binary complex E.NAD+, three forms of the ternary complex E.NAD+.alcohol, two forms of the ternary complex E.NADH.aldehyde and three binary complexes E.NADH. Appropriate pKa values have been ascribed to protonation forms of most of the above mentioned complexes of yeast enzyme with coenzymes and substrates.

Interactions of inhibitors at the coenzyme binding site of 6-phosphogluconate dehydrogenase☆

Abstract Yeast 6-phosphogluconate dehydrogenase was observed to be inhibited in a coenzymecompetitive fashion by a variety of nucleotides showing some degree of structural analogy to NADP + . The presence of a phosphate group at the 2′-position of the ribose moiety of a number of nucleoside derivatives studied provided a greater than expected stabilization of the binding of such compounds to this enzyme. N 1 -Alkylnicotinamide chlorides, as structural analogs of the pyridinium moiety of NADP + , were not effective inhibitors of this enzyme. Studies of the inactivation of yeast 6-phosphogluconate dehydrogenase by N -alkylmaleimides of varying chainlength indicated no positive chainlength effects in this inactivation process. The substrate, 6-phosphogluconate, protected the enzyme against inactivation by N -ethylmaleimide and this protection could be significantly increased by ternary complex formation with coenzyme or a coenzyme analog.

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.

Bull semen nicotinamide adenine dinucleotide nucleosidase: IV. Nonpolar interactions of inhibitors with the substrate binding site

Abstract NAD nucleosidase from bull semen was shown to be inhibited by a homologous series of aliphatic carboxylic acids, butanoic acid to decanoic acid, inclusive. The inhibition obtained was observed to be competitive with respect to NAD. The effectiveness of inhibition by these carboxylic acids increased with increasing chainlength of the alkyl groups of the inhibitors. n -Alkylphosphates, n -butylphosphate to n -dodecylphosphate, inclusive, were also found to inhibit NADase competitively. A positive chain-length effect was also observed in the binding of n -alkylphosphates to the enzyme. Interactions of these two groups of inhibitors with a pyrophosphate region at the substrate-binding site of the enzyme was suggested by the competitive nature of the inhibition obtained and by multiple inhibition analyses. Chain-length effects in the binding of these inhibitors of NADase suggest nonpolar interactions with a hydrophobic region of the enzyme to be of importance in these binding processes.

Self-inactivation of an erythrocyte NAD glycohydrolase.

SummaryNAD glycohydrolase activity was studied using bovine erythrocytes, erythrocyte ghosts and partially purified enzyme preparations. During catalysis the enzyme becomes irreversibly inactivated in a process related to substrate turnover. Self-inactivation was observed with intact cells, ghosts and solubilized enzyme and could be demonstrated with NAD, NADP and nicotinamide 1,N6 ethenoadenine dinucleotide as substrates. Thionicotinamide adenine dinucleotide and NADH, which are not substrates for the enzyme, do not inactivate but are reversible substrate-competitive inhibitors. Added thiols had no effect on enzyme self-inactivation. Of the reaction products, added nicotinamide partially protected the enzyme while added ADPR had no effect. Thermodynamic parameters calculated from Arrhenius plots for rate constants of self-inactivation indicate a large negative ΔS for transition state formation suggesting a process other than extensive denaturation. Erythrocyte ghost NADases from several other mammalian sources have been demonstrated to undergo a self-inactivation similar to that observed with the bovine enzyme.

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 rat ovarian 20α-hydroxysteroid dehydrogenase

Abstract Rat ovarian 20α-hydroxysteroid dehydrogenase was purified 230-fold with a 48% recovery through a 3-step process involving hydrophobic, gel filtration and gree dye affinity chromatography. The purified enzyme was demonstrated to be a single polypeptide chain of Mr 36 000. Initial velocity studies of all four substrates in the forward and reverse reactions indicated a sequential mechanism for the enzyme. Product inhibition and dead-end inhibition studies with substrate analogs were consistent with an ordered bi-bi mechanism in which NADP is the first substrate bound to the enzyme and NADPH, the second product released, Several NADP analogs were demonstrated to function as coenzymes in the reaction catalyzed. The purified enzyme was denatured at moderate temperatures and the binding of NADP protected the enzyme against thermal denaturation.