Catalytic zinc atom

The UV spectra of these complexes are very similar to those found in tetrahedral complexes of Co11 known to have a somewhat distorted geometry, suggesting a similar geometry in the cobalt enzyme.1383 Tetrahedral mercaptide complexes of the type [Co(SPh)4]2- were also shown to have similar absorption characteristics to those of [(LADH)Co2Co2j. This work is in complete agreement with the X-ray crystallographic studies of the native LADH, already mentioned, which shows distorted tetrahedral coordination of both the catalytic and non-catalytic zinc atoms of the enzyme. 

Fig. 25. Chain trace of one subunit of horse liver alcohol dehydrogenase. Stereo drawing from the work of Branden and colleagues [117], The catalytic zinc atom is central, the structural zinc atom is at the bottom right.

Fig. 26. (A) Schematic diagram of one subunit of horse liver alcohol dehydrogenase. Znl is the active-site zinc. Designed by B. Furugren, from the work of Branden and colleagues [55], (B) Schematic diagram of a section through the horse liver alcohol dehydrogenase dimer. The catalytic zinc atoms are shown, with the inhibitory substrate analogue DMSO and coenzyme molecules indicated. The dimer has two active sites, each composed of parts of both subunits. From the work of Branden and colleagues [123].

Of recent interest have been structural data on a novel class of MMP-binding inhibitors, represented by PNU-107859 (13) and PNU-142372 (14), which contain a thiadiazole moiety that coordinates the catalytic zinc atom through its exocyclic sulfur atom (130). 

Figure 3. The observed fold for astacin. Stereo ribbon [54] drawing of the observed structure of astacin [13], The catalytic zinc atom (cyan sphere) and the zinc binding histidine residues (blue) of the consensus sequence HexxHxxgxxH are identified. The structural elements defining the conserved zinc-endo-protease fold are shown in orange.

Figure 4. Comparison of astacin and the catalytic domain of coUagenase. Stereo ribbon diagram [54] of the structure of astacin (green) and the catalytic domain of coUagenase (orange) superimposed using a rigid body fit of the catalytic hehces. The catalytic zinc atom (cyan) and the zinc ligands (blue) are marked. The overall similarity in the open beta sandwich region, N-terminal to the active site helix is apparent.

Figure 7. The structure of thermolysin. Ribbon representation of the structure of thermolysin (silver, Brookhaven Databank [53] code 3TLN) shown with a bound inhibitor (green). The catalytic zinc atom (cyan) and structural calcium atoms (magenta) are shown. The active site is located between the N-terminal zinc protease domain and the alpha helical C-terminal domain. Zinc binding residues are in blue and the residue assisting catalysis is shown in red.

Figure 10. Comparison of the details of inhibitor binding in the two families superposition of the catalytic centers of collagenase (Fig. 6) and thermolysin (Fig. 9). The catalytic zinc atom is shown as a cyan sphere, with the collagenase-in-hibitor complex in yellow and the thermolysin complex shown in magenta. Thin cylinders denote putative hydrogen bonds. The green sphere shows the location of a structural calcium atom observed in collagenase. For clarity, only the alpha-carbon atoms of the proteins are shown. The differences in the relative oreintation of the subsites are evident.

Fig. 12. Stereo diagram of the active site pocket with the catalytic zinc atom.

The presence of two zinc atoms per subunit of LADH was first established by Akeson (134). He also showed that one zinc was essential for activity and suggested that the second zinc may have a structural role. There is no difference in zinc content between the multiple forms of the EE isozymes (164). Attempts to differentiate the function and chemical reactivities of the two zinc atoms have been described in a series of papers (133,165-167) which have been reviewed (168). One zinc atom per subunit could be selectively exchanged and removed by dialysis. The modified enzyme containing one zinc atom per subunit was catalytically inactive and did not bind 1,10-phenanthroline. From this information, combined with the X-ray data which show that the catalytic zinc atom binds 1,10-phenanthroline, it is evident that the catalytic zinc atom is first removed during dialysis under these conditions (167). The second zinc atom can be selectively removed, in preference to the catalytic zinc, by carboxymethylation (165). From sedimentation experiments of zinc-free... 

Kinetic and spectroscopic measurements support the hypothesis that the substrate binds directly to the catalytic zinc atom. Dunn and Hutchison (336) have produced evidence using a chromophoric aldehyde as substrate that the carbonyl oxygen of the reaction intermediate is coordinated to zinc. They concluded that zinc acts as a Lewis acid catalyst. Similar conclusions have been reached by McFarland and co-workers from the spectral properties of the enzyme complex with 4- (2 -imidazolyl-azo) benzaldehyde (337), from the observed small electronic substituent effect of para-substituted benzaldehydes (335), and from the absence of a large pH effect in the hydride transfer step (338). Assuming the mechanisms and the subunit structures to be essentially similar in YADH and LADH, a magnetic resonance study of coenzyme and substrate binding to YADH (339) also support the hypothesis of direct binding of substrate to zinc. 

Fig. 19. Schematic diagram illustrating the assumed positions of substrate and nicotinamide in relation to the catalytic zinc atom.

The results of cysteine modification confirm the similarities in structure and function of the active sites of mammalian and yeast alcohol dehydrogenases (Section II,D). Minor differences are, however, observed. Thus, the nicotinamide-substituted imidazole dinucleotide (137) selectively alkylates one of the two cysteine ligands to the catalytic zinc atom, Cys-43, in the yeast enzyme. In the horse enzyme, on the other hand, the same reagent alkylates a different ligand to the same zinc atom, Cys-174. 

The chelating agent 1,10-phenanthroline which binds to the catalytic zinc atom in LADH (Section II,C,3,b) is also an inhibitor for YADH 1,245,437,436,440,441)- However, the inhibitory power is considerably smaller with YADH than LADH )- Binding to zinc has been questioned 436) since nonchelating analogs such as 1,5-phenanthroline and... 

The structural and catalytic zinc atoms have also been replaced by cadmium-109 by equilibrium dialysis methods similar to that for cobalt, again either replacing just the structure zinc atoms giving [(LADH)Cd2Zn2], or both types of zinc giving [(LADH)Cd2Cd2]. The enzymic activity of both species was similar to the native enzyme. The cadmium-substituted enzyme is similar in all other respects to the cobalt substituted enzyme, i.e. mode of inhibition, UV spectra. The CdNMR spectrum has been observed for the Cd fully substituted enzyme and, as expected, shows two resonances corresponding to the two types of cadmium atoms in the enzyme. ... 

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