Thio-NAD

Occasionally, one can increase the Ae by utilizing alternative substrates. For example, 3-acetyl-NAD or thio-NAD can often be used with NAD -dependent dehydrogenases. Note however that an alternative substrate may change the kinetic mechanism, as compared to that observed with the naturally occurring substrate. Alternative substrates are of particular value when the normal substrate(s) and product(s) do not efficiently absorb UV or visible light. For example, many p-nitroaniline or p-nitrophenyl derivatives have proved to be quite useful in enzyme assays because they exhibit intense absorption around 410 nm. 

Substrates used were NADPH plus thio-NAD+. 

In order to measure transhydrogenation spectrophotometrically without a regenerating system, coenzyme analogs, e.g., thio-NAD(P) (absorption maximum for the reduced form at 400 nm), have often been used (6, 20). However, coenzyme analogs are not natural substrates and therefore care should be taken in interpreting kinetic data obtained with these artificial substrates. On the other hand, the use of analogs appears to be the method of choice in cases where rapid reactions are measured or where low concentrations of products interfere (see 21). 

Fig. 1. Prosthetic groups in oxidases (A FAD B Thio-Tyrosine C NAD(P) + D 6-Hydroxy-DOPA E Methoxanthin (Pyrroloquinoline quinone PQQ) F Tryptophane-Tryptophan quinone)...

Reduced forms of these coenzymes absorb ultraviolet light near 340 nm, whereas the oxidized forms do not. For NAD at neutral pH, the maximal absorbance band (s = 18000 M cm ) occurs at 260 nm another absorbance band (s = 8000 M cm ) occurs at 230 nm. For NADH, the maximal absorbance band (s = 16900 M cm ) occurs at 259 nm a second absorbance band (s = 6220 M cm ) occurs at 339 nm two weaker bands occur at 234 and 290 nm with respective s values of 6600 M cm and 1300 M cm h The same is true for NADP and NADPH at 339 nm. Occasionally, investigators have used thio-NADH which has a much stronger absorbance around 366 nm. See Absorption Spectroscopy (Fig. 4, pg. 5) Nicotinic Acid Analogs and Coenzymes... 

The behavior of invertebrate and plant GDH s has been less extensively studied than that of the bovine enzyme. The question of compulsory order as opposed to random order binding, which has been resolved only with great difficulty for bovine GDH, has been investigated with only a few other GDH s. In each case, for Phycomycetes GDH (NAD) (S3S), GDH (NADP) of Brevibacterium flavum 30), soybean GDH (NAD), 7), and both the NAD- and NADP-dependent GDH s of Thio-bacillus novellm (33), compulsory order binding has been reported in which coenzyme binds first and NH4+ last. However, since the more refined methods employed for investigation of the mechanism of bovine GDH have not been applied to any of these systems, the question of random vs. ordered mechanism cannot be said to have been resolved, particularly since the methods thus far employed did not give decisive results with bovine GDH. 

When Rudolph and Fromm used thionicotinamide adenine dinucleotide (thio-DPN) as an alternate substrate for NAD+ and varied the concentration of ethanol with liver alcohol dehydrogenase [following the reaction at 342 nm, the isosbes-tic point for thio-DPN and reduced thio-DPN (thio-DPNH)], they saw what appeared to be concave upward reciprocal plots with partial substrate inhibition in the presence of thio-DPN (38). However, the asymptote intercepts appeared to decrease with increased thio-DPN concentration, which is not what the above equations predict for a case where a minimum is present in the curve. There must have been other interactions that caused the substrate inhibition by ethanol in the presence of thio-DPN. 

The Michaelis constants are similar (2 X 10 Af for inosinate 0.2 to 4 X 10 M for NAD+) for enzymes from different sources, with the exception of Sarcoma 180 tumor cells, where that for NAD+ is 5 X 10 M 25). Both the AerobcuAer aerogenes enzyme 26) and that from Sarcoma 180 tumor cells 26) have been shown to catalyze strictly ordered reactions in which inosinate and xanthylate bind to free enzyme. The order of binding of other substrates (including H2O and NAD+) could not be determined. The phosphate of inosinate is important for binding of this substrate hence the dissociation constants of the 5 -phosphorothioate, 5 -thio, and 5 -amino analogues of inosinate are higher than that of inosinate 27, 28). 

Intimate Ion Pair Intermediates in the Solvolysis of Thio Addition Products of NAD(P) Analogs and Their Relevance to the Chemistry of 3-Phosphoglyceraldehyde Dehydrogenase... 

The three branched chain amino acids are normally metabolized as shown in Figure 6.2. Each amino acid is converted to the corresponding Of-keto acid by a transaminase specific for that amino acid. A solitary case of valinaemia is known, caused by lack of valine transaminase [76] the patient is mentally retarded. The three a-keto acids are decarboxylated by two (or possibly three) enzyme systems, one specific for a-keto-isovaleric acid, the other acting on a-keto-isocaproic and Q -keto-j3-methylvaleric acids [77, 78]. The reaction is complex, proceeding in three distinct steps [78] and requiring coenzyme A, thiamine pyrophosphate, lipoic acid and NAD. The end products are the co-enzyme A thio-esters of the branched chain fatty acids. 

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