Thermolysin-inhibitor complexes

Protease 3D structural models thermolysin-inhibitor complex ACE inhibitor drug design... 

The structure of thermolysin-inhibitor complexes has shown the presence of a deep PI specificity pocket and the details of hydrogen bonding between enzyme and inhibitor [28]. Side-chain atoms of the enzyme are involved in interaction with the inhibitor amides and the inhibitor conformation is such that the subsites PI and P2 both point into the enzyme core (Rg-9). 

Figure 9. Thermolysin-inhibitor complex - an example of inhibitors bound to zinc endoproteases with the long spacer consensus. A stereo view of the active site site of thermolysin showing details of enzyme-inhibitor 2 interactions (Brookhaven Databank Code 5TLN). Enzyme side chains involved in inhibitor recognition are shown in magenta. The color code is as given for Fig. 6.

Tronrud, D.E., Holden, H.M. and Matthews, B.W, (1987) Structures of Two Thermolysin Inhibitor Complexes that Differ by a Single Hydrogen Bond, Science, 235, 571-574. 

Free Energy Calculations on Enzyme-Inhibitor Complexes Studies of Thermolysin and Rhizopus Pepsin... 

In thermolysin (Matthews et al., 1974 Holmes and Matthews, 1982) zinc is bound approximately tetrahedrally to glutamate (monodentate), two histidines, and water. While zinc in the native enzyme is tetracoord-inate, in some inhibitor complexes it is pentacoordinate. The four calcium ions are bound by six to eight oxygen ligands, as shown in Fig. 35, with Ca 0 distances of 2.23-2.71 A. The RNA polymerase from E. coli contains Zn(II) and Mg(II), which may be substituted by Mn(II) (Chuk-nyhVy et al., 1990). 

The X-ray structures of enzyme and enzyme-inhibitor complexes permit the anhydride intermediate only in the case of carboxypeptidase, not in the case of thermolysin, since in this enzyme the catalytic Glu-143 is too far away from the substrate carbonyl (Lipscomb, 1983). The proposal that carboxypeptidase works via an anhydride intermediate thus requires the supposition that two very similar enzymes work by different mechanisms. 

Wasserman and Hodge (290) used molecular dynamics to dock thermolysin inhibitors to an approximate model of the enzyme, with flexibility in the active site (3 8 of 314 residues) and ligand and with the rest of the enzyme represented by a grid approximation. A solvation model was used to compensate for desolvation in complex formation. To get 22 of 25 runs to orient the hydroxamate function correctly, the hydroxamate oxygens of the starting conformation were initialized within 4 A of the zinc. If they were allowed to vary to 8 A, then only 3 of 24 runs placed the ligand correctly. Obviously, there is a serious sampling problem. 

Retro-inversion of hydroxamate enkephalinase inhibitors [89] leads to a 5-fold difference in potency between the (/ ) and (5) isomer compared with the 100-fold difference obtained with retrothiorphan. This difference between the thiol and hydroxamate inhibitors may be explained in terms of the geometrical parameters existing in their inhibitor-zinc complexes since in the thermolysin-thiol inhibitor complex, the zinc is tetra-coordinated [87], whereas in the thermolysin-hydroxamic acid inhibitor complex it is penta-coordinated [88]. The conformational space accessible to the thiol function is less than that accessible to the hydroxamate function, as there is only one degree of freedom in retrothiorphan (a rotation around the alpha... 

Thermolysin (TEN EC 3.4.24.28), a thermostable bacterial protease isolated from Bacillus thermoproteolyticus, has been studied as the prototype of zinc-metallopeptidases at a time where no crystal structure was available for this class of proteases [122]. Crystallographic analysis of a number of TLN/inhibitor complexes has allowed an understanding of the binding mode of these inhibitors and allowed the mechanism of action of this protease to be determined [122]. These seminal studies have greatly inspired the development of NEP inhibitors, given the close stmctural relationship between TEN and NEP [123]. To examine further the structural relationships between these two peptidases, various phosphinic peptides were prepared. One of these compounds (58, Table 1) exhibits a Ki value of 26 nM toward thermolysin and 22 nM toward NEP [124]. 

Figure 1. Ribbon diagram of thermolysin complexed the inhibitor Cbz-GlyP-NH-Leu-Leu (stick) and Zn (sphere) in the active site (PDB code 5TMN).

Any examination of crystal structures of complexes of a series of ligands binding to a protein (the set of complexes of thermolysin with a variety of inhibitors determined in the Brian Mathews lab, for example see references in DePriest et al. [36]) shows clearly a major limitation of the pharmacophore assumption. Ligands do not optimize overlap of similar chemical functionality in complexes but find a way to maintain correct hydrogenbonding geometry, for example, while accommodating other molecular interactions. 

Fig. 3. Structure of the inhibitor phosphoramidon. The tetrahedral phosphorus atom binds at the active site of thermolysin and mimics the transition-state complex.

Figure 8 shows the MCD spectra for these two complexes. The spectra of the model complexes resemble well the Co2+-thermolysin spectra. Substitution of 2-methylimidazole for imidazole produces a spectrum similar to that for the Co2+-thermolysin-/J-phenylpropionyl-6-phenylalanine complex, while the spectrum with imidazole resembles closely that of the Co2+-thermolysin spectrum without inhibitor. 

Bash et al. (1987) applied the thermodynamic perturbation method to complexes of thermolysin with a phosphonamidate [Cbz-Gly -(NH)-Leu-Leu] and the corresponding phosphonate inhibitor [Cbz-Gly -(0)-Leu-Leu]. The perturbation was carried out by using 20 windows, with 2-psec molecular dynamics simulations in each window. Computations were for the ligand in solution and bound to the enzyme. The solvation of the enzyme was represented by a spherical cap of 168 water molecules about the bound inhibitor. The difference in free energy of binding of the two inhibitors was calculated to be 4.38 kcal/mol, to be compared with the experimental value, 4.10 kcal/mol. These calculations point out the importance of solvation effects, which are seen in the 3.4 kcal/mol difference between the NH and O forms of the inhibitor. 

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