Activity Measurements The activity of prolyl oligopeptidase was determined fluorometrically with Z-Gly-Pro-Nap (Bachem, Ltd.), using a Jasco FP 777 spectrofluorometer. The excitation and emission wavelengths were 340 nm (1.5 nm bandwidth) and 410 nm (5 nm bandwidth), respectively. The substrate with internally quenched fluorescence, Abz-Gly-Phe-Gly-Pro-Phe-Gly-Phe(NO 2)-Ala-NH 2, was prepared with solid phase synthesis, and its hydrolysis was followed similarly as in the case of Z-Gly-Pro-Nap, except that the excitation and emission wavelengths were 337 and 420 nm, respectively. Kinetics The specificity rate constants ( k cat /K m) were determined under first-order conditions, i.e. At substrate concentrations lower than K m.

The first-order rate constant, calculated by nonlinear regression analysis, was divided by the total enzyme concentration to provide k cat /K m. The pH dependence of catalysis was measured in a four-component buffer composed of 25 m m glycine, 25 m m acetic acid, 25 m m Mes, and 75 m m Tris and which contained 1 m m EDTA and 1 m m 1,4-dithiothreitol (standard buffer).

The buffer was titrated to the desired pH with HCl or NaOH, while the ionic strength remained fairly constant over a wide pH range. Small changes in the conductivity were adjusted by the addition of NaCl. After the reaction had been completed, the pH of each sample was determined and found to be practically identical with the starting value. Theoretical curves for bell-shaped pH rate profiles were calculated by nonlinear regression analysis, using the following equation Equation 1and the GraFit software (). In Equation k stands for k cat/ K m, and (limit) refers to the pH independent maximum rate constant. K 1 and K 2 are the dissociation constants of a catalytically competent base and acid, respectively. The pH rate profiles composed of two bell-shaped curves were fitted to the following equation Equation 2 where k(limit) 1 and k(limit) 2 gave the limiting values of the rate constant for the low pH and high pH forms of the enzyme.

The K i values, the dissociation constant of the enzyme-inhibitor complex, were calculated from the following equation Equation 3where k i and k 0 are pseudo first-order rate constants determined at substrate concentrations at least 10-fold less than K m in the presence and absence of inhibitor (I), respectively. An additional important feature of substrate binding concerns the P2 carbonyl oxygen, which forms two hydrogen bonds, one with Arg 643 as already pointed out (), and the other with the leaving group (NH of the P1′ residue). These hydrogen bonds appear to stabilize the substrate in the proper position for catalysis and explain an earlier observation that a sulfur atom in place of the P2 carbonyl oxygen makes the substrate practically unsuitable for hydrolysis (). The sulfur substitution of the carbonyl group in the P2 position seems to induce a greater effect on catalysis than does such a substitution in the P1 position, where the carbonyl oxygen is directly involved in the peptide bond cleavage (). An extensive network of hydrogen bonds (P2′-NHHis 680-Nε2, P1′-NHP2-COArg 643-Nη1P4-CO) has not been observed with other serine peptidases. In summary, the unique intramolecular hydrogen bond (P1′-NHP2-CO) can possibly help the general acid catalysis, i.e. Chota Bheem 3gp Video Download In Hindi.

The proton transfer from the imidazolium ion to the leaving group. Binding of a Product-like Inhibitor It has previously been shown that the acyl product of Z-Gly-Pro-Nap, the classic substrate of prolyl oligopeptidase, is an inhibitor to the enzyme (). B illustrates that the binding mode of Z-Gly-Pro-OH greatly resembles the complex formed between prolyl oligopeptidase and the aldehyde inhibitor Z-Pro-prolinal (Fig.

Similarity to the P1-P3 residues of the octapeptide binding (Fig. A) is also apparent, and the interactions are listed in Table. One of the oxygen atoms of the carboxylate ion of Z-Gly-Pro-OH is located in the oxyanion binding site, forming hydrogen bonds with the OH group of Tyr 473 and the main chain NH of Asn 555. The other oxygen atom is linked to the Nε2 of His 680.