NAD complexes

The active center of an LDH subunit is shown schematically in Fig. 2. The peptide backbone is shown as a light blue tube. Also shown are the substrate lactate (red), the coenzyme NAD (yellow), and three amino acid side chains (Arg-109, Arg-171, and His-195 green), which are directly involved in the catalysis. A peptide loop (pink) formed by amino acid residues 98-111 is also shown. In the absence of substrate and coenzyme, this partial structure is open and allows access to the substrate binding site (not shown). In the enzyme lactate NAD"" complex shown, the peptide loop closes the active center. The catalytic cycle of lactate dehydrogenase is discussed on the next page. 

The evidence available is consistent with mechanism A depicted in Fig. 3. Oxidation of the a-D-glucopyranosyl pyrophosphate derivative (107a) at C-4" by NAD is followed by irreversible elimination of water from the /3-hydroxy ketone 109, with subsequent reduction of the conjugated ketone (110) by NADH. Only the NAD complexes can release the sugar nucleotide, and, consequently, the intermediates 109 and 110 are not present in free form, uncom-plexed with enzyme-NADH. 

The ADP produced by the hydrolysis of ATP is continuously used up by added purified pyruvate kinase, which in the presence of phosphoenol pyruvate produces pyruvate and ATP (Fig. 3.8.6). Pyruvate is then utilized by added lactate dehydrogenase, which in the presence of NADH produces lactate and NAD+. Complex V activity is estimated from the rate of NADH oxidation at 340 nm (e 4870-M 1-cm 1 isosbestic point 380 nm), after subtracting the oligomycin-resistant activity. It should be kept in mind that oligomicyn sensitivity requires the preserved attachment of the Fr component of the enzyme to the membranous F0 component. The attachment is readily lost upon freeze-thaw cycles. Consequently, it is reasonable to measure the activity on fresh material only. 

Both these mechanisms propose that the alcohol substrate combines with the unprotonated form of the enzyme-NAD+ complex. Kvassman and Pettersson have proposed an alternative mechanism in which alcohol binding to the binary complex requires the presence of a neutral... 

The steady state and stopped-flow kinetic studies on the horse liver enzyme are now considered classic experiments. They have shown that the oxidation of alcohols is an ordered mechanism, with the coenzyme binding first and the dissociation of the enzyme-NADH complex being rate-determining.15,26,27 Both the transient state and steady state methods have detected that the initially formed enzyme-NAD+ complex isomerizes to a second complex 27,28 In the reverse reaction, the reduction of aromatic aldehydes involves rate-determining dissociation of the enzyme-alcohol complex,27,29 whereas the reduction of acetaldehyde is... 

In this reaction, NAD must bind first to yield the E-NAD complex to which malate then combines to form a ternary complex, E-NAD-malate the main reaction then takes place to yield NADH and oxaloacetate. 

A clue to the physico-chemical basis of the correlation comes from the observation from the crystal structures of various dehydrogenase-NAD+ complexes that those enzymes which bind NADH in the syrt-conformation [45a] transfer the pro-5 hydrogen whereas those enzymes that bind NADH in the a // -conformation [46b] transfer the pro-/ hydrogen (see e.g. You, 1984). The observed stereospecificities thus correspond to transfer of hydride towards the viewer from both rotamers of [45]... 

Pure crystalline GAPDH has been isolated from a number of different sources (cf. Table I) (7, 8,10, H-28). Methods of purification have relied heavily upon its solubility as the enzyme-NAD complex in high concen-... 

Michaelis constants of the non-energy-linked beef heart transhydrogenase reaction are 9 ftM for NADH, 40 nM for NADP , 28 )tM for NAD , and 20 pM for NADPH (68) these values are similar to those found with other AB-specific transhydrogenases (66, 89 see also 8). Dissociation constants for the E-NADH and E-NAD complexes, derived from... 

Protection of the reductase by NADH from inhibition by thiol group reagents suggested that the enzyme formed a stable complex with pyridine nucleotide (353). Such a complex was readily demonstrated by difference spectroscopy. When the enzyme was reduced by NADH or AcPyADH a prominent positive band was observed at 317 nm (Fig. 16) this band was very small (and blue-shifted) when NADPH, a very poor substrate, was the reductant. Furthermore, addition of NAD+ following NADPH resulted in a difference spectrum identical with that produced by NADH. The dashed line in Fig. 15 represents the absorption resulting from NAD" binding. Thus, this band was attributed to a reduced enzyme-NAD complex (350). 

All these structures also contained a disordered region in the vicinity of the active site, but the amount of disorder was substantially less than that seen in the original structure. While residues 352-390 are still disordered in apo yMIPS, the C2 yMIPS/NAD+ complex, and three of the four molecules of the P2i NAD+-bound yMIPS complex, (315 residues (391-408) are relatively well-ordered in most of these structures. A smaller still disordered region is reported in the P2j2j2 yMIPS/NAD+ crystal form. In this structure, only residues 365-375, encompassing a 14 are disordered (Kniewel et al., 2002). 

Figure 7. Space filling model of yMIPS/NAD+ complex. NAD+ atoms are darkened relative to the protein atoms.

The Z isomer of the model substrate 4-tra s- N,]V -dimethylamino)cinnamaldoxime (Z-DMOX) forms a ternary complex with NAD and LADH. The Co", Ni, Cu and Cd enzymes (with the metal substituted for the catalytic zinc) and the apoenzyme also form ternary complexes with Z-DMOX. The affinity of the apoenzyme-NAD complex for Z-DMOX is much lower and the rate of Z-DMOX dissociation from the apoenzyme complex was 10 -fold greater than the rates found for the metal-substituted enzymes. Complex formation results in a red shift (43 to 83.5 nm) in the DMOX UV-visible spectrum, due, it is suggested, to bonding of the oxime nitrogen to a strong electrophilic centre, either the Zn or the nicotinamide ring of NAD, a view that is compatible with the known structural features of the LADH/NADH/MejSO complex. The high affinities and slow rates of dissociation of the metal-substituted enzyme complexes are attributed to the coordination of Z-DMOX to the active site metal. ... 

Dutler and Branden (11, 12) have studied the interaction of the ADH-NAD complex with alkyl-substituted cyclohexanols to determine productive substrate orientations in the active site. Some of their findings were that 1) cyclohexanol fits best in a chair conformation with axial-reacting hydroxyl oxygen and equatorial-reacting hydrogen atoms on Cl, 2) that substitutions at Ck (para) have little effect on the reaction rate due to hydrophobic bonding with the residues in the hydrophobic "barrel region of the active site, 3) substitutions at C2,... 

Dehydrogenase enzymes that use NAD+ as a cofactor follow this mechanism, since an enzyme-NAD+ complex forms initially, and changes the local structure at the enzyme s active site to allow substrate binding. 

Figure 16.4-3. Aldehyde dismutase acitivity of Thermoanaerobium brockii alcohol dehydrogenase (TBADH). A high affinity of the TBADH-NAD+ complex for hydrated acetaldehyde is proposed, explaining the stochiometric acetaldehyde dismutation.

The dismutation reaction has important practical implications. The coupling of the thermodynamically favorable oxidation of a secondary alcohol to drive the reduction of an aldehyde is a common strategy for dehydrogenase-mediated specific reductions in bioreactors. These conditions, however, are ideal for dismutation of the aldehyde. As shown in Fig. 17, the Enz-NAD+ complex is... 

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