Chymotrypsin active enzyme dynamics

A reaction looked at earlier simulates borate inhibition of serine proteinases.33 Resorufin acetate (234) is proposed as an attractive substrate to use with chymotrypsin since the absorbance of the product is several times more intense than that formed when the more usual p-nitrophcnyl acetate is used as a substrate. The steady-state values are the same for the two substrates, which is expected if the slow deacylation step involves a common intermediate. Experiments show that the acetate can bind to chymotrypsin other than at the active site.210 Brownian dynamics simulations of the encounter kinetics between the active site of an acetylcholinesterase and a charged substrate together with ah initio quantum chemical calculations using the 3-21G set to probe the transformation of the Michaelis complex into a covalently bound tetrahedral intermediate have been carried out.211 The Glu 199 residue located near the enzyme active triad boosts acetylcholinesterase activity by increasing the encounter rate due to the favourable modification of the electric field inside the enzyme and by stabilization of the TS for the first chemical step of catalysis.211... 

Very fast electron transfers from P+ to bacteriochlorophyl (Bchl) and from (Bchl)- to QA do not depend on media dynamics and occur via conformationally non-equilibrium states (Fig.3.18). The dual fluorophore-nitroxide molecules (D-A) are also convenient objects for analysing the activity-dynamics relationship. The marked irreversible photoreduction of the nitroxide fragment of the dual probe incorporated into the binding site of HSA only took place when the nanosecond dynamical processes around the probe traced by ESR and fluorescence methods were detected (Rubtsova et al., 1993, Fogel et al, 1994 Likhtenshtein, 1986 Lozinsky et al., 2002). Similar results were reported for another model protein system, i.e. a-chymotrypsin with spin labeled methionin-92 groups (Belonogova et al., 1997). In the latter enzyme, the excited tryptophan group serves as an electron donor. 

We report the results from a molecular dynamics simulation of the serine protease y-chymotrypsin (y-CT) in hexane. The active site of chymotrypsin contains the "catalytic triad" which consists of Ser-His-Asp. y-CT suspended in nearly anhydrous solvents has been found to be catalytically active. In order for proteins to retain their activity in anhydrous solvents some water molecules are required to be present. These "essential waters" have been suggested to function as a molecular lubricant for the protein. Hexane, having a dielectric constant of 1.89, is a suitable non-aqueous solvent for enzymatic reactions. The low dielectric constant of hexane allows it to not compete with the protein for the essential water and allows enzymes to retain their catalytic activity. y-CT in hexane is thus an ideal system to further explore the effect of non-aqueous solvation on protein structure, function and dynamics. 

Experiments performed in a very similar way also showed dynamic disorder for the enzyme a-chymotrypsin [16], a-Chymotrypsin is an endopeptidase acting on water-soluble polypeptides. The substrate (suc-AAPF)2-rhodamine 110 was designed to interact optimally with the binding site of the enzyme. It consists of a rhodamine 110 core that is derivatized with a succiny-lated AlaAlaProPhe peptide sequence, known to bind very specifically at the enzyme s active site (Fig. 25.2a). To further avoid potential artifacts, the enzyme was immobilized by entrapment in an agarose matrix, which restricts enzyme diffusion while still allowing free rotation and conformational dynamics of the enzyme as well as the diffusion of the substrate. 

In a similar manner, Latif et al. [104] utilized MD simulations in order to gain insight into the structural properties and dynamics of a-chymotrypsin in imidazo-lium-based ILs with different types of anions ([bmim]PF, [bmim]BF, [bmim]Cl, [bmim]TfO, and [bmim]NTf2). At a low water content, the conformation of enzyme was closer to its native structure in the presence of ILs, presenting also a bell-shaped dependency on water content. However, no major conformational changes at the active site of the enzyme were observed. The solvation of the enzyme in water-immiscible ILs led to a higher enzyme flexibility at increased water content. 

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