Transition state structure determination: analysis of the hydration of 2-acetyl-3,4-dimethylthiazolium ion and. cap alpha. -chymotrypsin catalyzed hydrolyses using the proton inventory technique
The equilibrium hydration of 2-acetyl-3,4-dimethylthazolium ion was investigated in water-acetonitrile mixtures by using the proton inventory technique and by measuring thermodynamic activation parameters. The solvent isotope effect, k/sub H/sub 2/O/k/sub D/sub 2/O/, on the rate constant, k/sub f/, for the forward step of the equilibrium hydration varied from 3.10 to 2.58 as the volume fraction of acetonitrile was increased from 0.25 to 0.90. The solvent isotope effects were found to be consistent with a transition state consisting of two water molecules in which one water molecule acts as a general base to abstract a proton from the nucleophilic water molecule. The transition state was found to be invariant within this range of solvent composition. The kinetics of the deacylation step of the ..cap alpha..-chymotrypsin and 1-methyl-His-57 chymotrypsin catalyzed hydrolysis of p-nitrophenyl acetate, p-nitrophenylthiolacetate, N-Cbz-glycine-p-nitrophenyl ester, and N-Cbz-glycly-glycine-p-nitrophenyl ester and the native ..cap alpha..-chymotrypsin catalyzed hydrolysis of the minimal substrates p-nitrophenyl acetate and p-nitrophenylthiolacetate were investigated by the proton inventory techniques. These results were interpreted in terms of a mechanism involving initial acylation of the imidazole ring of His-57 with the substrates p-nitrophenyl acetate and p-nitrophenylthiolacetate, and by a mechanism involving the unmethylated nitrogen of His-57 acting as a general base in catalysis of the amino substrates by methylchymotrypsin.
- Research Organization:
- Texas A and M Univ., College Station (USA)
- OSTI ID:
- 7002696
- Country of Publication:
- United States
- Language:
- English
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