Coenzyme binding domain

Ohlsson, I., Nordstrom, B., Branden, C.-I. Structural and functional similarities within the coenzyme binding domains of dehydrogenases. /. Mol. Biol. 89 339-354, 1974. 

Figure 4.9 (a) Triose phosphate isomerase (TIM), has a (3-a-(3 structure made up of eight P-a motifs terminating in a final a-helix, which form a barrel-like structure, (b) An open twisted P-sheet with helices on both sides, such as the coenzyme-binding domain of many dehydrogenases. (From Branden and Tooze, 1991. Reproduced by permission of Garland Publishing, Inc.)... 

A crystal structure of a ternary complex of horse liver alcohol dehydrogenase with NADH and the inhibitor, dimethyl sulfoxide, first at 4.5 A resolution1365 and a further refinement to 2.9 A resolution,1366 has been published by Eklund et al. The gross structure of the ternary complex is similar to that of the free enzyme structure. Each subunit is divided into a coenzyme-binding domain and a catalytic domain. The subunits are joined together near the... 

Certain combinations of secondary superstructures are often found in proteins and control their structure and function. The most frequent is the /fayS-unit, where an a-helix bridges two /1-strands. This is the prevailing feature in most coenzyme-binding domains of dehydrogenases [7]. Other important superstructures include a,a-dimers, /1-meanders and //-barrels. 

The observation of at least two transitions is likely not due to the presence of a protein contaminant Our gel electrophoresis analysis of sodium dodecyl sulfate-treated YADH yielded only one band that corresponded to the molecular weight of a YADH subunit (10), Thus, the presence of labile species or domain(s) in the sample must account for the two transitions. For example, the low Tm transition could be attributed to the denaturation of a labile dissociated subunit or a labile domain in the tetramer. Such labile subunits or domains may be akin to the conformationally drifted species found for other dehydrogenases (2,5). The high Tm transition, in turn, may be attributable to the denaturation of an active tetramer, or coenzyme binding domain, because the transition exhibited the greatest response to NAD. Other alternatives are that tetrameric YADH reversibly dissociates and the subunits are responsible for the low Tm transition, or that the low and high Tm transitions can be attributed to thermally-induced dissociation and denaturation of the subunits, respectively. To aid the interpretation of the scans, additional DSC experiments were performed. 

The reaction involves the hydride transfer from the substrate to the pyridine C-4 position of NAD(P)+. This transfer is usually stereospecific, being the oxidoreductase either anti or syn, depending on the rotation by 180° of the nicotinamide ring with respect to the ribose moiety. During the catalytic cycle, when formed, NAD(P)H dissociates and is replaced by an incoming NAD(P)+ indicating that NAD(P)H exhibits a weaker enzyme affinity. An oxidoreductase usually consists of two domains, a coenzyme-binding domain, like the Rossmann fold (8, 9), and an adjacent catalytic domain where the substrate binds (Fig. 2). 

Liver alcohol dehydrogenase subunit viewed as a CPK model. The left-hand side of the molecule is the coenzyme binding domain and the right-hand side is the catalytic domain. The catalytic zinc ion is accessible from two channels located above (not visible) and below the coenzyme binding domain. The upper channel permits approach of the nicotinamide ring of the coenzyme. The lower channel permits approach of the substrate. The substrate channel closes up, trapping the substrate inside the molecule, when both coenzyme and substrate are present. 

A structure such as the six-stranded coenzyme binding domain in the dehydrogenases would be disrupted by insertions or deletions of amino acids (see Fig. 7 for elaboration). Hence, sequence comparisons of parallel pleated sheet regions are particularly reliable. Structural methods of alignment of sheet areas have been discussed in Section II. The corresponding amino acid comparisons are made in Table IV. For tbe purpose of this chapter, the present LDH numbering scheme (4) will be used as the generalized reference system. 

The structural studies have thus shown for all dehydrogenases investigated that (a) the folding of the coenzyme binding domains are similar,... 

Subunit differences have also been characterized. One of the amino acid differences between two types of subunits is a Val/Ala exchange (73) at position 43 (with the numbering of the horse enzyme, Table I). The subunit responsible for the atypical human alcohol dehydrogenase has been reported to have Pro instead of Ala (75) at position 230. This position is, in the horse enzyme, in aC in the coenzyme binding domain (Fig. 

Each subunit is clearly divided into two domains joined by a narrow neck region and separated by the deep active site cleft. One of these domains, called the coenzyme binding domain, binds the coenzyme. The two zinc atoms are bound within the second domain, called the catalytic domain. The two domains are unequal in size the catalytic domain is larger and comprises 231 residues, whereas the coenzyme binding domain is built up from 143 residues. The two subunits of the dimeric molecule... 

SI, 9II, and /Sill are the three pleated sheet regions of the catalytic domain schematically illustrated in Fig. 10. The individual strands of these sheets are numbered sequentially from the amino terminal, dk. .. 0F are the strands of the parallel pleated sheet region in the coenzyme binding domain schematically illustrated in Fig. 5. The helices are labeled al. . . a4 in the catalytic domain and oA. . . E in the coenzyme binding domain. Ill. . . R17 are reverse bends defined according to Venkatachalam 117). 

Fig. 3. Stereo diagram of the positions of the a-carbon atoms of the coenzyme binding domain in LADH and the bound ADP-ribose molecule.

Fig. 4. The main chain hydrogen bonding pattern of the residues involved in the coenzyme binding domain of LADH.

Fig. 5. Schematic diagram illustrating the fold of the polypeptide chain within the coenzyme binding domain and the nomenclature of the elements of secondary structure.

Residues in the Coenzyme Binding Domain Which Form Hydrophobic Cores ... 

Both subunits contribute residues to each active site pocket. The bottom part of the pocket which binds zinc, nicotinamide, and the reactive part of the substrate is contained entirely within each subunit. The second half of the pocket, closer to the surface, however, is lined by residues from the catalytic domain of one subunit and from the region 3 S of the coenzyme binding domain of the other subunit. 

A. Thus, there is no direct interaction between the two catalytic centers. Indirect ihteraction mediated by the conformational change induced by the coenzyme is, however, quite possible, especially since the subunits are bound together through their coenzyme binding domains. 

---