Hydrolases transition state

Schowen, R. L. 2003 Biochemistry 42, 1900—1909 The catalytic strategy of S-adenosyl-L-homocysteine hydrolase Transition-state stabilization and the avoidance of abortive reactions. 

The mechanism for the lipase-catalyzed reaction of an acid derivative with a nucleophile (alcohol, amine, or thiol) is known as a serine hydrolase mechanism (Scheme 7.2). The active site of the enzyme is constituted by a catalytic triad (serine, aspartic, and histidine residues). The serine residue accepts the acyl group of the ester, leading to an acyl-enzyme activated intermediate. This acyl-enzyme intermediate reacts with the nucleophile, an amine or ammonia in this case, to yield the final amide product and leading to the free biocatalyst, which can enter again into the catalytic cycle. A histidine residue, activated by an aspartate side chain, is responsible for the proton transference necessary for the catalysis. Another important factor is that the oxyanion hole, formed by different residues, is able to stabilize the negatively charged oxygen present in both the transition state and the tetrahedral intermediate. 

Fig. 3.6. Stereoelectronic control of the cleavage of the tetrahedral intermediate during hydrolysis of a peptide bond by a serine hydrolase. The thin lines represent the reactive groups of the enzyme (serine, imidazole ring of histidine) the thick lines represent the tetrahedral intermediate of the transition state. The full circles are O-atoms open circles are N-atoms. The dotted lines represent H-bonds the thick double arrow indicates an unfavorable dipole-dipole interaction [21]. A (R)-configured N-center B (S)-configured N-center.

General acid catalysis is schematized in Fig. 7J,b. Here, an acid A-H increases the polarity of the carbonyl group and, hence, the electrophilicity of the carbonyl C-atom. For entropy reasons, the reaction is greatly facilitated when it is an intramolecular one (Fig. 7J,b2), in other words, when the general acid catalyst is favorably positioned within the molecule itself. Such a mechanism is the one exploited and refined by nature during the evolution of the hydrolases, with the general acid catalyst and the H20 molecule replaced by adequate amino acid side chains, and the enzymatic transition state being de facto a supermolecule (see Chapt. 3). 

Fig. 10.6. Simplified representation of the postulated catalytic cycle of microsomal and cytosolic epoxide hydrolases, showing the roles played by the catalytic triad (i.e., nucleophile, general base, and charge relay acid) and some other residues, a) Nucleophilic attack of the substrate to form a /3-hydroxyalkyl ester intermediate, b) Nucleophilic attack of the /Thydroxyal-kyl ester by an activated H20 molecule, c) Tetrahedral transition state in the hydrolysis of the /f-hydroxyalkyl ester, d) Product liberation, with the enzyme poised for a further catalytic... 

W. Nerinckx, T. Desmet, K. Piens, and M. Claeyssens, An elaboration on the syn-anti proton donor concept of glycoside hydrolases. Electrostatic stabilisation of the transition state as a general strategy,... 

M. Degano, S. C. Almo, J. C. Sacchettini, and V. L. Schramm, Trypanosomal nucleoside hydrolase. A novel mechanism from the structure with a transition-state inhibitor, Biochemistry, 37 (1998) 6277-6285. 

M.-C. Ho, W. Shi, A. Rinaldo-Matthis, P. C. Tyler, G. B. Evans, K. Clinch, S. C. Almo, and V. L. Schramm, Four generations of transition-state analogues for human purine nucleoside hydrolase, Proc. Natl. Acad. Sci. USA, 107 (2010) 4805 -812. 

Furneaux RH, Limberg G, Tyler PC, Schramm VL (1997) Synthesis of transition state inhibitors for N-riboside hydrolases and transferases. Tetrahedron 53 2915-2930... 

Horenstein BA, Parkin DW, Estupinan B, Schramm VL (1991) Transition-state analysis of nucleoside hydrolase from Crithidia fasciculate. Biochemistry 30 10788-10795... 

Horenstein BA, Schramm VL (1993) ElecUonic nature of the transition state for nucleoside hydrolase. A blueprint for inhibitor design. Biochemistry 32 7089-7097... 

Ema T, Jittani M, Furuie K, Utaka M, Sakai T (2002) 5-[4-(l-Hydroxyethyl)phenyl]-10,15,20-triphenylporphyrin as a probe of the transition-state conformation in hydrolase-catalyzed enantioselective transesterifications. J Org Chem 67 2144-2151... 

Several 0-GlcNAcase inhibitors have been described, the most popular are streptozotocin (STZ) and PUGNAc. Both of these compounds are substrate mimetics that inhibit 0-GlcNAcase activity by resembling the natural substrate s ox-azoline transition state after its entrance into the active site (50, 51). Unfortunately, the above inhibitors are nonselective and can inhibit other glycosyl hydrolases therefore, they are a detriment to a multitude of cell pathways. Another, more potent transition state analog, A-acetylglucosamine-thiazoline (NAG-thiazoline), has been described recently (5). The increased potency likely is because the compound already resembles the oxazoline intermediate before it is exposed to the enzyme. Macauley et al. 

The concept of electrostatic catalysis has been applied indirectly by Schramm and coworkers for the design of transition-state analogues in reactions by nucleoside hydrolase (Horenstein et al., 1991 Horenstein and Schramm, 1993) and AMP deaminase (Kline and Schramm, 1994). They stated, in accordance with our previous examples, that the MEP provides novel insight into transition-state structure and the forces associated with substrate binding and release. 

Nucleoside hydrolase has been proposed to participate in purine salvage in the trypanosome Crithidia fasciculata. The enzyme hydrolyses the N-glycosidic linkage of the naturally occurring purine and pyrimidine nucleosides. A geometric model of the transition state for nucleoside hydrolase for the reaction (Horenstein et al., 1991)... 

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