NAD+ binding

Fig. 12.1. Domain scheme of selected proteins with internal ubiquitin-like domains. Ubiquitin-like domains are indicated by black boxes. Other domains are abbreviated as follows ThiF, NAD-binding domain in ubiq-uitin activating enzymes UAct, 2nd conserved domain in ubiquitin activating enzymes ...

Glyceraldehyde-3-phosphate dehydrogenase is a homotetramer that carries out the oxidative phosphorylation of glyceraldehyde-3-phosphate into 1,3-bisphos- phoglycerate. During this reaction NADH is formed. Each subunit of the enzyme consists of two domains and has an NAD+ binding site. The N-terminal domain anchors the adenosine portion of the cofactor while the nicotinamide portion is involved in the catalytic reaction at the C-terminal domain. T brucei... 

Stereoview of NAD binding by trypanosomal GAPDH (full black). For clarity only 2 of the 4 subunits are shown. A substantial deviation of the protein backbone occurs in human GAPDH (open gray, only one subunit shown) near the adenosine part of the cofactor. The nearby cleft (marked by an asterisk) is important for introducing selectivity in inhibitor binding and has therefore been termed selectivity cleft. ... 

RGURE 13-16 The nucleotide binding domain of the enzyme lactate dehydrogenase, (a) The Rossmann fold is a structural motif found in the NAD-binding site of many dehydrogenases It consists of a six-stranded parallel /3 sheet and four a helices inspection reveals the arrangement to be a pair of structurally similar motifs... 

The surface plasmon resonance minimum reflectivity in Figure 20-23 shifts by —0.15° when 1 mM NAD+ binds to the imprinted polymer. The shift is not as great for the related species NADH, NADP+, and NADPH, confirming that the imprinted polymer selectively binds NAD+. When the observed reflectivity was fitted to the theoretical response, the polymer film was calculated to be 22 3 nm thick and had a binding capacity of 2.26 xg NAD+/cm2. When 1 mM NAD+ binds to the polymer, the refractive index of the polymer layer changes from 1.45 to 1.40 and the layer thickness increases by 3.0 0.2 nm. 

Figure 15-10 The three-dimensional structure of glutathione reductase. Bound FAD is shown. NAD+ binds to a separate domain below the FAD. The two cysteine residues forming the reducible disulfide loop are indicated by dots. From Thieme et al.182...

Figure 7.4 Activation of PARP-1 by DNA breaks. PARP-1 is composed of three domains, DNA binding, automodification and catalytic (NAD+ binding) domains (1). In cells, PARP-1 localizes to nucleoli and actively transcribed regions of chromatin by interacting with RNA. When PARP-1 binds to DNA breaks, PARP-1 initiates the poly(ADP-ribosyl)ation reaction by using NAD+ as its substrate (2). PARP-1 itself is the main target of the poly(ADP-ribosyl)ation reaction. ADP-ribose polymers are formed on the automodification domain of PARP-1 (automodification). As a consequence of automodification, PARP-1 dissociates from DNA breaks (3). When cells are committed to apoptosis, PARP-1 is specifically cleaved by an apoptosisspecilic protease, caspase-3, resulting in the formation of a 24kDa N-terminal and 89 kDa C-terminal fragments (4). (see Color Plate 7)...

The color code provides information about the similarity of amino acids, and the boxes give examples of information gathering from the multiple alignment the first box shows the FAD binding domain, the second box shows a highly conserved cysteine residue crucial for this family, and the third box shows a consensus as part of the NAD binding domain. 

Nearly all NAD+-dependent dehydrogenases studied follow an ordered bisubstrate mechanism. In this mechanism, the oxidation of a substrate proceeds in a sequential manner first, NAD+ binds in the active site of the dehydrogenase then the substrate binds next a hydride equivalent is transferred in a chemical step from the bound substrate to the bound NAD+, hence, oxidising the substrate and reducing the NAD+ to NADH the oxidised substrate is then released from the active site and is finally followed by the NADH. 

Fig. 3. A hypothetical ribozyme that can catalyze electron transfer. Aptamers than can bind NAD+ (and, hence, NADH) are selected, and the binding domain is mapped. An oligonucleotide tail that can bind to an unpaired region near the NAD-binding domain is attached to FMN. The bound FMN-oligonucleotide will be adjacent to NADH when it is bound in the active site of the ribozyme. Electron transfer should occur owing to the proximity of the two substrates. The rate of the reaction can be controlled by varying the length of the oligonucleotide tail to vary the distance between NADH and FMN substrate. Although this catalyst is extremely simple (and employs the same principles of catalysis found in nonenzymatic template-directed ligation reactions), it would nevertheless demonstrate the ability of RNA to catalyze reactions other than phosphodiester bond transfers.

A domain is an independently folded region of a protein e.g., the NAD+-binding domain of glyceraldehyde-3-phosphate dehydrogenase. 

Fig. 33), giving the required topological features between strands 4 and 1, which is where the pyrophosphate moiety of NAD binds [72]. 

Fig. 33. Diagram illustrating the arrangement of strands and helices in the NAD-binding domain of dehydrogenases. (B designed by B. Furugren.) From the work of Branden and colleagues [72],...

Fig. 3. Stereoviews of the Co atom backbone in lobster muscle GAPDH (a) one subunit viewed to illustrate the NAD -binding and catalytic domains (b).the NAD -binding domain viewed in the same orientation as in (a) (c) the catalytic...

Flo. 4. Diagrammatic representation of the NAD -binding domain showing the six-stranded parallel p sheet flanked by helices (4S, 6S). 

Fig. 5. The main chain hydrogen bonding scheme in the NAD -binding domain (residues 1-149K4S, S3).

Fig. 6. Stereoview of the NAD -binding site showing amino acid side chains interacting with the coenzyme. Note Phe-34 and Phe-99 on either side of the adenine ring. Aspartate-32 and Gly-7, which are close to the adenine ribose, preserve then-functions in other dehydrogenases (48, S3).

Correlation of conserved and variable regions with the three-dimensional structure (S9) shows that residues involved in catalysis and in intersubunit contacts are conserved to a much greater extent than others. It follows that the sequence of the catalytic domain with its greater proportion of active site and subunit contact residues is more highly conserved than the sequence of the NAD-binding domain despite the fact that the latter represents a highly conserved structure. 

The early studies of Velick et al. (134) first delineated the two aspects of NAD binding that have provided the basis for subsequent work. These... 

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