Enzymes entatic state

A hypothetical conformational state defined by a geometrically and electronically strained site within an enzyme thought to facilitate the conversion of an enzyme-substrate complex to the transition state. Vallee and Williams defined the entatic state as an abnormal condition of localized strain transmitted by relief of compression or other steric clashes elsewhere in the enzyme. They suggested that catalytic rate enhancements arise from the heightened reactivity of catalytic group(s) that have experienced unimolecular activation. Williams ... 

Table I lists isomorphous replacements for various metalloproteins. Consider zinc enzymes, most of which contain the metal ion firmly bound. The diamagnetic, colorless zinc atom contributes very little to those physical properties that can be used to study the enzymes. Thus it has become conventional to replace this metal by a different metal that has the required physical properties (see below) and as far as is possible maintains the same activity. Although this aim may be achieved to a high degree of approximation [e.g., replacement of zinc by cobalt(II)], no such replacement is ever exact. This stresses the extreme degree of biological specificity. The action of an enzyme depends precisely on the exact metal it uses, stressing again the peculiarity of biological action associated with the idiosyncratic nature of active sites. (The entatic state of the metal ion is an essential part of this peculiarity.) Despite this specificity, the replacement method has provided a wealth of information about proteins that could not have been obtained by other methods. Clearly, there will often be a compromise in the choice of replacement. Even isomorphous replacement that should retain structure will not necessarily retain activity at all. However, it has become clear that substitutions can be made for structural studies where the substituted protein is inactive (e.g., in the copper proteins and the iron-sulfur proteins). It is also possible to substitute into metal coenzymes. Many studies have been reported of the...

There is a third region of a protein that is neither on the surface nor in the interior but that is in a cleft. Such regions are often associated with enzyme action and examples show they have (1) intermediate mobility (e.g., tryptophan 62 of lysozyme or the tyrosine of carboxypeptidase) and (2) unfavorable energetics of exposed groups—the entatic state. 

I have a question for Professor Williams regarding the concept of entasis [B. L. Vallee and R. J. P. Williams, Proc. Nat. Acad. Sci. (U.S.) 59, 498 (1968)]. Is it possible that many enzymes in structured regimes in vivo exist in an entatic state, and that such states collapse (e.g., the protein folds up) on extraction from the cell ... 

If our postulates are correct the most interesting feature of P-450 is the manner in which the protein has adjusted the coordination geometry of the iron and then provided near-neighbour reactive groups to take advantage of the activation generated by the curious coordination. Vallee and Williams (68) have observed this situation in zinc, copper and iron enzymes and referred to it as an entatic state of the protein. It is also apparent that some such adjustment of the coordination of cobalt occurs in the vitamin B12 dependent enzymes. As a final example we have looked at the absorption spectra of chlorophyll for its spectrum is in many respects very like that of a metal-porphyrin. This last note is intended to stress the features of chlorophyll chemistry which parallel those of P-450. 

Each molecule (molecular weight 30000) contains one zinc(II) atom, which is (approximately) tetrahedrally coordinated to two N atoms and one O atom from amino-acid residues plus a water molecule. The structures of both the enzyme and some enzyme-substrate complexes have been carefully studied and the detailed mechanism of the hydrolysis is now quite well understood. Without going into details, a crucial factor appears to be the pronounced distortion from regular tetrahedral coordination about the Zn(II), apparently imposed by the conformational requirements of the polypeptide chain. The conflict of interest between the needs of the Zn(II) atom - which, when four-coordinate, always assumes tetrahedral coordination - and the ligands induces an entatic state, a condition of strain and tension which enhances the reactivity at the active site. The Zn atom binds the substrate peptide via the O atom of the —CONH— peptide link, and the entatic state of the free enzyme facilitates formation of the enzyme-substrate complex. 

Enzymes are Nature s catalysts, facilitating all of the chemical reactions of metabolism. They bind a specific substrate in an entatic state moving it along the reaction coordinate towards the reaction transition state thus lowering the reaction activation energy. Enzymes are generally made of proteins and contain an active site, often based around a metal ion. 

Figure 3 A typical metallo-enzyme, azurin. The copper ion in it is a constrained (entatic) state matching its function. The copper in the enzyme is not open to any substrate—it is an electron-transfer protein. See Reference 8.

The additional comment that the high aflSnity of metalloenzymes for their metals as "compared with the stability of chelates which use the same ligands, argues against a thermodynamically strained coordination is similarly not relevant and based upon a misinterpretation of the entatic site hypothesis. Entasis implies that the difference in energy between the ground state and transition state for the enzymatic reaction is reduced, not that the metal-enzyme complex is thermodynamically less stable, as was inferred. Indeed, there is no reason to suppose that the distorted environment of a metal ion in an enzyme as opposed to a simple metal complex leads necessarily to an increase in free energy. The studies of alkaline phosphatase just presented certainly seem consistent with the entatic state hypothesis. 

The visible and near-i.r. spectral properties of the enzymatically active cobalt(ii) carboxypeptidase have been examined and it is concluded that the co-ordination geometry of the metal atom at the active site is a distorted tetrahedron (in agreement with the m.c.d. results ). Consideration of the magnetic susceptibility of this enzyme, on the other hand, suggests that the metal has a five-co-ordinate geometry whereas that in the corresponding nickel enzyme, which is also active, is octahedral. It is difficult to reconcile this result with the entatic state concept of catalytic activity. 

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