Interactive computer graphics was used as a tool in studying the cleavage mechanism of the model substrate Z-Phe-Phe-Leu-Trp by the zinc endopeptidase thermolysin. Two Michaelis complexes and three binding orientations of the tetrahedral intermediate to the crystal structure of thermolysin were investigated. Our results indicate that a Michaelis complex, which does not involve coordination of the scissile peptide to the zinc, is consistent with available experimental data and the most plausible of the two complexes. A tetrahedral intermediate complex wherein the two oxygens of the hydrated scissile peptide straddle the zinc in a bidentate fashion results in the most favorable interactions with the active site. The preferred tetrahedral intermediate and Michaelis complex provide a rationalization for the published substrate data. A trajectory for proceeding from the Michaelis complex to the tetrahedral intermediate is proposed. This trajectory involves a simultaneous activation of the zinc-bound water molecule concurrent with attack on the scissile peptide. A detailed ordered product release mechanism is also presented. These studies suggest some modifications and a number of extensions to the mechanism proposed earlier [Kester, W. R., & Matthews, B. W. (1977) Biochemistry 16, 2506; Holmes, M. A., & Matthews, B. W. (1981) Biochemistry 20, 6912]. The binding mode of the thermolysin inhibitor N-(1-carboxy-3-phenylpropyl)-L-leucyl-L-tryptophan [Monzingo, A. F., & Matthews, B. W. (1984) Biochemistry (preceding paper in this issue)] is compared with that of the preferred tetrahedral intermediate, providing insight into this inhibitor design.

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Dedicated to Professor Jack D. Dunitz on the occasion of his 90th birthdayThe hydrophobic effect is viewed as the driving force for the aggregation of nonpolar substances with extended lipophilic molecular surfaces in aqueous solution through the exclusion of water molecules from the formed interfaces. [1,2] It is usually quoted to explain why an oil/water mixture spontaneously separates, why soluble proteins fold with a hydrophobic core and a hydrophilic outer surface, [3,4] why membrane components assemble as lipid bilayers and micelles, why membrane proteins are accommodated in membrane segments, and why small molecules associate in protein binding pockets with mutual burial of hydrophobic surfaces. [5] In the latter instance, it is a general strategy in medicinal chemistry to improve protein-ligand binding by increasing the ligands hydrophobic surface which becomes buried in hydrophobic pockets of the target protein. In all cases, the hydrophobic effect is considered to be the major force of association. On the molecular level, this phenomenon is commonly attributed to the displacement of water molecules arranged around the hydrophobic surfaces, and entropic effects are made responsible to drive this association. The entropic profile is related to changes in the degree of ordering and the dynamic properties of the water molecules, which are assumed to be more disordered in the bulk water phase relative to where they were located prior to being displaced upon hydrophobic association. Recent studies have demonstrated, however, that hydrophobic interactions can originate either from enthalpyor entropy-driven binding, making simple explanations often presented for the hydrophobic effect insufficient. [6][7][8][9][10][11][12] Also in computational design tools the handling of explicit water molecules has received increasing recognition. Tools such as WaterMap and Szmap [13,14] try to take into account water structures in drug design and the properties of individual water molecules are discussed in terms of enthalpy and entropy.In order to obtain a better understanding of the hydrophobic effect on the molecular level and its role in proteinligand binding, we embarked on a systematic study using thermolysin (TLN) as a model system. [6] This thermostable bacterial zinc metalloprotease from Bacillus thermoproteolyticus exhibits three specificity pockets of predominantly hydrophobic nature (Scheme 1). It has been considered a prototype for the entire class of enzymes [15] owing to its highly conserved active-site architecture, despite remarkable sequence differences to other zinc proteases. Potent TLN inhibitors are often designed as transition-state analogues. [16][17][18] The enzyme has been frequently used as a surrogate [19][20][21][22][23] for other metalloenzymes against which new drugs are developed, and served as a model system to test ideas [24,25] and new methodological concepts. [26] TLN was one of the first crystallographically investigated metalloproteases [27,28] and its catalytic zinc ion is coordinated ...

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