Kinetics and mechanism of trypsin catalysis.
Creators: name: Kasserra, H. P.
Publisher: name: Université d'Ottawa / University of Ottawa
Year: 1969
Subjects: subject: Chemistry, Organic.

description: A steady-state kinetic study has been made of the trypsin-catalyzed hydrolysis of N-benzoyl-L-alanine methyl ester, at pH values ranging from 6-10. From the rates were calculated, at each pH values of k˜c/K˜m, k˜c and K˜m (=k˜c(k˜-1+k˜2)/k˜ 1k˜2). The pH profiles of these parameters provide pK values for the groups that ionize in the free enzyme and in the acylenzyme. The kinetic results on the acid side imply that there is a group of pK &amp;sime; 7 in trypsin, presumably the imidazole function of a histidine residue, and that this group is involved in acylation and deacylation, both of which can only occur if it is unprotonated. The behaviour on the basic side revealed a decrease in kc at high pH corresponding to a value of pK &amp;sime; 9.5, whereas k˜ c/K˜m showed sigmoid pH dependence. This pK was related to the alpha-amino group of the N-terminal isoleucine residue. The specific levorotation of trypsin and trypsinogen has been measured as a function of pH over the pH range 5-11. The change in specific rotation of trypsin follows the ionization of a single group with a pK(app) of 9.4 which was not revealed by the corresponding curve for trypsinogen. At pH 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 pH-dependence curves of the specific rotation of trypsin in the presence of reversible competitive inhibitors were found to be displaced to a more alkaline pH. This was interpreted to indicate a conformational stabilization as the result of enzyme-inhibitor complex formation with a consequent increase in the pK(app) of the group revealed by the free enzyme. An interpretation of the kinetic and optical rotary results that is consistent with all available information is that the group of pK &amp;sime; 9.5 (presumably the -NH3+ function of the terminal isoleucine residue) controls the conformation and thereby the activity of the enzyme in alkaline solution. The transient-phase kinetics of the trypsin-catalyzed hydrolysis of N-carbobenzoxy-L-alanine-p-nitro-phenyl ester have been studied using a stopped-flow technique. Under conditions of excess enzyme concentration, the pH-dependencies of k˜2 and K˜ m (=k˜-1+k˜2)/k˜1) have been obtained without the need to make any assumptions as to the relative magnitude of k˜3. From the results it was concluded that a group of pK = 6.9 participates in acylation and that the same group is not involved in Michaelis-complex formation. On the basis of the steady-state work, this group has already been postulated to be the imidazole function of the histidine residue. A least-squares-fit computer program was developed to analyze the kinetic rate measurements directly and was used in place of the traditionally employed Guggenheim method. The effect of organic solvent on the pre-steady state parameters has been investigated for solvent concentrations varying from 1-20% (v/v) isopropyl alcohol. The changes in k˜2, K˜m and k˜ 2/K˜m could be formally correlated with the reciprocal of the dielectric constant of the solvent mixture, indicating an increase in electrostatic interactions. It was concluded that the observed decrease in both binding and the rate of hydrolysis of N-carbobenzoxy-L-alanine-p-nitrophenyl ester can be justified by a loss in conformational integrity of the enzyme. This is the result of increased free-energy contributions due to electrostatic and hydrophobic interactions with little or no effect due to hydrogen bonding. The chemical and mechanistic evidence for reactions catalyzed by trypsin and chymotrypsin has been reviewed in some detail and is considered with reference to the structure of chymotrypsin as determined by X-ray studies. Conclusions have been drawn about the detailed nature of the processes of Michaelis-complex formation, acylation and deacylation which are incorporated in a concerted reaction mechanism. Although there are significant differences between the two enzymes as far as specificity and pH effects are concerned, it has been concluded that their mechanism of catalysis is the same.