The stopped-flow technique has been used to study the pre-steady-state kinetics of the hydrolysis of N-carbobenzoxy-L-alanine-p-nitrophenyl ester catalyzed by trypsin. By working under conditions such chat the enzyme concentration is much greater than that of the substrate, it has been possible to measure k2, the rate constant for the conversion of the enzyme-substrate complex into the acyl enzyme. The p H dependence of 4, reveals a pKbl value of 6.9 for the conversion of complex into acyl enzyme, in agreement with deductions from steady-state investigations. The pHdependence of R* (equal to (k-, + k2)/k,) has also been determined. The results provide direct evidence for the existence of an enzyme-substrate complex for this reaction.The work has bzen done in various mixtures of water and isopropyl alcohol. The logarithms of the rate constants L2 and k2/R* vary linearly with 1/D, showing a decrease with increasing alcohol concentration;x* increases with alcohol concentration. The solvent results suggest that addition of alcohol affects the hydrophobic bonding in the protein and leads to unfolding of the enzyme.

A kinetic study has been made of the trypsin-catalyzed hydrolysis of N-benzoyl-L-alanine methyl ester, at p H values ranging from 6 to 10. The substrate concentrati~ns varied from 1.7 x to 4.3 x M. From the rates were calculated, at each pH, values of kc (corresponding to [S] >> K,,,), k,/f?,,,, (corresponding to [S] << K,,,) and K,,,. The specific levorotation of trypsin was measured and found to vary with p H in the p H region 5-1 1, the change in specific rotation following the ionization of a single group with pK(app) of 9.4. At p H 11 the specific rotation of trypsin, its zymogen, and its phosphorylated derivative were approximately the same, suggesting similar conformations for all three forms of the protein.The kinetic results on the acid side were very similar to those obtained by other investigators for chymotrypsin; they imply that there is a group of pK, z 7 in the free enzyme, presumably the imidazole function of a histidine residue, and that this group is involved in acylation and deacylation, which can only occur if it is unprotonated. The behavior on the basic side was found to be different from that with chymotrypsin revealing a decrease in kc at high p H corresponding to a value of pK, z 9.5, whereas kc/I?,,, showed sigmoid pH-dependence. A n interpretation of these results that is consistent with all available information is that a group of pK z 9.5 (presumably the -NH3+ function of the terminal isoleucine) controls the conformation and thereby the activity of the enzyme at different stages of complex formation. In contrast to chymotrypsin, the pK of this ionizing group appears to be generally lowered by covalent complex formation between trypsin and its substrates.

The mechanistic evidence for reactions catalyzed by chymotrypsin and trypsin is briefly reviewed and is considered with reference to the structure as determined by the X-ray studies. Conclusions are drawn about the detailed nature of the processes of complex formation, acylation, and deacylation. Although there are significant differences between the two enzymes as far as specificity and p H effects are

No abstract

The preparation of two new sulphonyl derivatives of methoxy-and naphtha-hydroquinone a s possible intermediate compounds in the synthesis of redox monomers is described. The standard oxidation potentials of these and other sulphonyl derivatives of hydroquinones were determined and compared with the values for similar compounds, reported previously (1).In Part I1 (I) we have described the preparation of redox polymers containing repeating hydroquinone units joined through a sulphone bridge to a polystyrene chain.Although 2,s-dihydroxydiphenyl sulphones were first synthesized by Hinsberg (2, 3) in 1894-1895, and in some cases their oxidized forins prepared, no systematic study of redox potentials of these and similar derivatives of hydro-and naphthahydro-quinones has been made. The sulphones whose redox potentials have been reported are p-toluenesulphonyl, p-bromobenzenesulphonyl, and d-camphor-10-sulphonyl derivatives of 1,4-naphthahydroquinone (4, 5) and the d-camphor-10-sulphonyl derivative of 2,s-dimethylhydroquinone (4). The redox potentials of these compounds are 121, 125, 114, and 135 mv, respectively, higher than the potentials of the corresponding parent quinones.Data previously obtained from potentiometric titrations (I) on intermediate and model cornpounds of redox polymers of the sulphone type showed the effect of various substituent 'Taken i n part from the thesis submitted by H. P . Kasserva