A plausible identification of secondary binding site in trypsin and trypsinogen

1. The determination of the binding of 4,4'diazoamino-bis-benzamidine (DABB) to alfa-trypsin by equilibrium measurements in columns indicated a stoichiometry of 2 mol ligan/mol enzyme. One molecule binds to the secondy binding site, sith Ki2=mMat pH8,0, 25-C. 2. Bovine pancreatic trypsin inhibitor (BPTI) prevented binding of DABB to both sites, indicating that they are topographically close and within the interface of the trypsin-BPTI complex. 3. On the basis of data from the interface of the trypsin-BPTI complex, we concluded that the secondary binding site of trypsin is plausibly identified as the same site in trypsin that binds the Arg-17 reside of BPTI, i.e., Tyr-39 and Tyr-151 in bovine trypsin. This site would then correspond to subsite S'2 on the enzyme surface

A statistical treatment for solution of a family of simultaneous equations derived from enzyme inhibition studies

1. A techinique of simultaneous regression of a family of lines with any common intercept is described for the treatment of enzyme inhibition data. The method is iterative and is baed on minimiztion of a weighted of squares of all residues. 2. The weighting procedure may be varied according to experimental design; the simultaneous regression treatment provides the crossing point of all linear and angular coefficients, as well as estimates of the precision of fitted parameters. 3. There are no restrictions as to location of crossing points. The method can be applied to all known transformed functions, properly weighted, used in enzyme studies that result in straight lines. It is rather simple to use by non-kineticists and would be useful in preliminary diagnosis of inhibition types, prior to the application of nonlinear techniques which require knowledge of the model

The substituent effect on complex formation between alfa-trypsin and para-substituted benzamidinium ions: A thermodynamic study

Dissociation constants, Kj, were determined spectrophotometrically by measuring the absorbance at 410 nm, using N alfa-benzoyl-D,L-argomome-para-nitroanilide (Bz-D,L-Arg-Nan) as substrate. The Ki values for the complexes of alfa-trypsin with each of the para-derivatives of the benzamidinium ion -NH2, -CH3, -H, -F, -Cl, -Br, -COOEt, and -NO2 were measured at six temperatures (8,15,20,25, 29 and 33§C), in order to determine the thermodynamic parameters for complex formation. The standard enthalpy change was constant and all other parameters were also negative. The large negative values obtained for the standard heat capacity change suggest that the process occurs with a conformational adaptation in the enzyme structure. The apparent partial specife volumes of free alfa-trypsin and alfa-trypsin bound to benzamidinium ion indicated that there is a decrease of approximatelly 0.10 cm**3/g in the enzyme volume when the inhibitor binds. This contraction is consistent with the release of about 130 water molecules per enzyme molecule