AdenineDinucleotide by KineticMethods (original) (raw)
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An Investigation of the Active Site of Lactate Dehydrogenase with NAD+ Analogues
European Journal of Biochemistry, 1981
The kinetic properties of 18 NAD' analogues, with alterations to the nicotinamide moiety, have been studied with respect to dogfish M4, rabbit M4 and beef H4 lactate dehydrogenases. The size of the groups present at C-3 of the pyridinium can be increased quite extensively without loss of coenzyme activity. Groups tested were thioamide, methyl, ethyl, diazoketone and chloroacetyl. Substitutions at positions C-4 and C-5 prevent proper positioning for hydride transfer and can hinder binding to the enzyme. The kinetic properties of pyridine-adenine dinucleotide and its 3-iodo derivative reveal the binding role of the amide at C-3 whereas 3-cyanopyridine-adenine dinculeotide is a strong inhibitor.
Evidence for binding of NAD dimers to NAD-dependent dehydrogenases
Biochimica et Biophysica Acta (BBA) - Enzymology, 1981
The binding of dimers of nicotinamide adenine dinucleotide, (NAD)2, to lactate, malate and alcohol dehydrogenase has been studied by the fluorescence quenching technique. While the alcohol dehydrogenase shows a low binding ability, malate and lactate dehydrogenases have been found to bind (NAD)2 in a specific way with high affinity. Malate dehydrogenase binds (NAD)2 more than NADH. All three dehydrogenases are inhibited by (NAD)2, which behaves as a competitive inhibitor with respect to both NAD ÷ and NADH. These results show that (NAD)2 is bound to the nucleotide-specific binding site of the dehydrogenases. (NAD)2 was found to stoichiometrically react with ferricyanide at variance with NADH. The specific interactions with the NAD-dependent dehydrogenases and the ability to enter in monoelectronic redox cycles suggest possible physiological roles for (NAD)z.
Effect of NADH-X on Cytosolic Glycerol-3-phosphate Dehydrogenase
Archives of Biochemistry and Biophysics, 1998
At pH 7.05 NADH-X prepared by incubating NADH with glyceraldehyde-3-phosphate dehydrogenase (E.C. 1.2.1.12) was a potent noncompetitive inhibitor, with respect to coenzyme, of NADPH oxidation by pure rabbit muscle cytosolic glycerol-3-phosphate dehydrogenase (E.C. 1.1.1.8) and also a potent inhibitor of NADPH oxidation catalyzed by this enzyme in a rat pancreatic islet cytosolic fraction. It was a much less potent inhibitor of NADPH oxidation catalyzed by this enzyme in a rat liver cytosolic fraction and of NADH oxidation catalyzed by this enzyme from all three sources. Glycerol-3-phosphate dehydrogenase purified from muscle cytosol contains tightly bound NADH-X, NAD, and ADP-ribose, each in amounts of about 0.1 mol per mole of enzyme polypeptide chain. A deproteinized supernatant of this enzyme contained these three ligands and produced the same type of inhibition of the enzyme described above for prepared NADH-X with a K i , in the reaction with NADPH at pH 7.05, in the range of 0.2 M with respect to the total concentration of ligands ([ADP-ribose] ؉ [NAD] ؉ [NADH-X] ؍ 0.2 M). However, only the NADH-X component could account for the potent inhibition because NAD, ADP-ribose, and the primary acid product (which can be produced from NADH-X) each had a K i considerably higher than 0.2 M. Although at pH 7.05 NADH-X inhibited NADPH oxidation considerably more than NADH oxidation, the reverse was the case at pH 7.38. Since the enzyme purified from muscle contains tightly bound NADH-X, NADH-X might become attached to the enzyme in vivo where it could play a role in regulating the ratio of NADH to NADPH oxidation of the enzyme.
Archives of Biochemistry and Biophysics, 1988
The 2',3'-dialdehyde nicotinamide ribose derivatives of NAD (oNAD) and NADH (oNADH) have been prepared enzymatically from the corresponding 2',3'-dialdehyde analogs of NADP and NADPH. Pig heart NAD-dependent isocitrate dehydrogenase requires NAD as coenzyme but binds NADPH, as well as NADH, ADP, and ATP, at regulatory sites. Incubation of l-3 InM oNAD or oNADH with this isocitrate dehydrogenase causes a time-dependent decrease in activity to a limiting value 40% that of the initial enzyme, suggesting that reaction does not occur at the catalytic coenzyme site. Upon varying the concentration of oNAD or oNADH from 0.2 to 3 mM, the inactivation rate constants increase in a nonlinear manner, consistent with reversible binding of oNAD and oNADH to the enzyme prior to covalent reaction. Inactivation is accompanied by incorporation of radioactive reagent with extrapolation to 0.54 mol [14C]oNAD or 0.45 mol ['4C]oNADH/mol average enzyme subunit (or about 2 mol reagent/m01 enzyme tetramer) when the enzyme is maximally inactivated; this value corresponds to the number of reversible binding sites for each of the natural ligands of isocitrate dehydrogenase. The protection against oNAD or oNADH inactivation by NADH, NADPH, and ADP (but not by isocitrate, NAD, or NADP) indicates that reaction occurs in the region of a nucleotide regulatory site. In contrast to the effects of oNAD and oNADH, oNADP and oNADPH cause total inactivation of the NAD-dependent isocitrate dehydrogenase, concomitant with incorporation, respectively, of about 3.5 mol [14C]oNADP or 1.3 mol [14C]oNADPH/mol average subunit. Reaction rates exhibit a linear dependence on [oNADP] or [oNADPH] and protection by natural ligands against inactivation is not striking. These results imply that oNADP and oNADPH are acting in this case as general chemical modifiers and indicate the importance of the free adenosine 2'-OH of oNAD and oNADH for specific labeling of the NAD-dependent isocitrate dehydrogenase. The new availability of 2',3'-dialdehyde nicotinamide ribose derivatives of NAD, NADH, NADP, and NADPH may allow selection of the appropriate reactive coenzyme analog for affinity labeling of a variety of dehydrogenases. o 1988 Academic press, fnc. Affinity labeling using nucleotide analogs has proved to be an effective means of accomplishing specific chemical modification of coenzyme and regulatory sites in proteins (1, 2). Of the classes of analogs used, the 2',3'-dialdehyde derivatives of nucleotides have been among the most 1 This work was supported by USPHS Grant DK39075. ' To whom correspondence should be addressed. widely employed. Periodate oxidation results in the cleavage of the bond between carbons 2' and 3' of the ribose leading to ready synthesis of the 2',3'-dialdehyde derivatives of adenine, guanine, uridine, and cytidine nucleotides (e.g., (3, 4)). As corn-. pared to the natural nucleotides, these compounds exhibit relatively minor changes in their structure but are capable of reacting covalently with amino acid side chains of enzymes. In the case of 665
Studies with Analogues of Nicotinamide Adenine Dinucleotide
European Journal of Biochemistry, 1974
Through a series of correlative studies, the stereochemistry of the enzymatic processes with the coenzyme analogues of NAD+, 3-acetylpyridine-adenine dinucleotide, thionicotinamide-adenine dinucleotide and 3-cyanopyridine-adenine dinucleotide, have been examined for horse liver and yeast alcohol dehydrogenase (both A-specific for NAD+ ) and glutamate dehydrogenase (Bspecific for NAD+). I n each case the stereochemistry of the process with respect to substrate and coenzyme remains identical to that for NAD+. The implications for this are examined in view of the different geometry of the substituents a t the 3-position of the pyridine ring. A ready conversion of the thionicotinamide analogue of NADH to NADH is also described.
European Journal of Biochemistry, 1993
Using a significantly simplified modification procedure, four charged analogues of the coenzyme NAD, N(1)-and W-(2-hydroxy-3-trimethylammoniumpropyl)-NAD, N(1)and N6-(3-sulfopropyl)-NAD were prepared. The kinetic parameters of these derivatives and N(1)-(2-aminoethyl)-NAD, W -(Zaminoethyl)-NAD and tricyclic 1 ,N6-ethanoadenine-NAD, all with alterations to the adenine moiety, were determined for porcine heart lactate dehydrogenase isoenzyme H,. The coenzyme activity depends on both position and charge of the introduced groups. Modification of the W-position leads to a 25 -250-fold increase of the k,,/K,,, value compared to the related N(1) derivative. The k,,/K,,, value for 1 ,W-ethanoadenine-NAD is in the range between that of N(1)-(2-aminoethyl)-NAD and W-(2-aminoethyl)-NAD. In the case of both N(1) and N6 functionalization, the K, values increase
NADH Dehydrogenases: From Basic Science to Biomedicine
Journal of Bioenergetics and Biomembranes, 2001
This review article is concerned with two on-going research projects in our laboratory, both of which are related to the study of the NADH dehydrogenase enzyme complexes in the respiratory chain. The goal of the first project is to decipher the structure and mechanism of action of the proton-translocating NADH-quinone oxidoreductase (NDH-1) from two bacteria, Paracoccus denitrificans and Thermus thermophilus HB-8. These microorganisms are of particular interest because of the close resemblance of the former (P. denitrificans) to a mammalian mitochondria, and because of the thermostability of the enzymes of the latter (T. thermophilus). The NDH-1 enzyme complex of these and other bacteria is composed of 13 to 14 unlike subunits and has a relatively simple structure relative to the mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I), which is composed of at least 42 different subunits. Therefore, the bacterial NDH-1 is believed to be a useful model for studying the mitochondrial complex I, which is understood to have the most intricate structure of all the membraneassociated enzyme complexes. Recently, the study of the NADH dehydrogenase complex has taken on new urgency as a result of reports that complex I defects are involved in many human mitochondrial diseases. Thus the goal of the second project is to develop possible gene therapies for mitochondrial diseases caused by complex I defects. This project involves attempting to repair complex I defects in the mammalian system using Saccharomyces cerevisiae NDI1 genes, which code for the internal, rotenone-insensitive NADH-quinone oxidoreductase. In this review, we will discuss our progress and the data generated by these two projects to date. In addition, background information and the significance of various approaches employed to pursue these research objectives will be described.
Stoichiometry of the nadh-oxidoreductase reaction for dehydrogenase determinations
Clinica Chimica Acta, 1980
The NADH oxidoreductase reaction with resazurin was most rapid at pH 6.5. FMN (10 pmol/l) markedly stimulated the reaction, and the optimal concentration of resazurin was 50 pmol/l. The oxidation of NADH by NADH oxidoreductase with resaruzin as electron acceptor gave a variable yield of fluorescent product, resorufin. The yield was pH dependent and was greatest at pH 6.5. Measurements of oxygen consumption in the reaction mixture demonstrated that dissolved O2 was an alternative electron acceptor. The increased yield of resorufin at pH 6.5 was due to more rapid reduction of resazurin rather than oxygen. In contrast, at pH 9.0 oxygen was the preferred electron acceptor. The sensitivity of assays utilizing this indicator reaction can be improved by these optimized conditions.