The α-chymotryptic hydrolysis of glycine esters

Ingles, D. W.; Knowles, J. R.

doi:10.1042/bj0990275

Cited by 24 publications

(9 citation statements)

References 17 publications

(8 reference statements)

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“…The plots fell on the same curve for initial enzyme concentrations of 3.2 and 6.4pM, indicating apparent first-order kinetics. These conditions of stirring in glass are similar to those of the pH-stat experiments of Hofstee (1965) and Ingles & Knowles (1966), where first-order kinetics were found.…”

Section: Adsorption Experiments and The Interpretation Of Kinetic Ressupporting

confidence: 79%

The effects of surface and macromolecular interactions on the kinetics of inactivation of trypsin and α-chymotrypsin

Johnson

Whateley

1981

Biochemical Journal

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The autolysis of trypsin and alpha-chymotrypsin is accelerated in the presence of colloidal silica and glass surfaces. It is proposed that adsorption of the enzymes (favoured by electrostatic factors) results in a conformational change that renders the adsorbed enzyme more susceptible to proteolytic attack. Although the adsorbed enzymes are more susceptible to proteolysis, their activity towards low-molecular-weight substrates is not affected, indicating a relatively minor conformational change on adsorption. The rates of autolysis in solution (i.e. in ;inert' vessels) are second-order for both trypsin and alpha -chymotrypsin, with rate constants of 13.0mol(-1).dm(3).s(-1) for trypsin (in 50mm-NaCl at pH8.0 at 25 degrees C) and 10.2mol(-1).dm(3).s(-1) for alpha-chymotrypsin (in 0.1m-glycine at pH9.2 at 30 degrees C). In glass vessels or in the presence of small areas of silica surface (as colloidal silica particles), the autolysis of both trypsin and alpha-chymotrypsin can show first-order kinetics. Under these conditions, saturation of the surface occurs and the fast surface proteolytic reaction controls the overall kinetic order. However, when greater areas of silica surface are present, saturation of the surface does not occur, and, since for a considerable portion of the adsorption isotherm the amount adsorbed is approximately proportional to the concentration in solution, second-order kinetics are again observed. A number of negatively charged macromolecules have been shown similarly to increase the rate of autolysis of trypsin: thus this effect, observed initially with glass and silica surfaces, is of more general occurrence when these enzymes adsorb on or interact with negatively charged surfaces and macromolecules. These observations explain the confusion in the literature with regard to the kinetics of autolysis of alpha-chymotrypsin, where first-order, second-order and intermediate kinetics have been reported. A further effect of glass surfaces and negatively charged macromolecules is to shift the pH-activity curve of trypsin to higher pH values, as a consequence of the effective decrease in pH in the ;microenvironment' of the enzyme associated with the negatively charged surface or macromolecule.

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Section: Adsorption Experiments and The Interpretation Of Kinetic Ressupporting

confidence: 79%

The effects of surface and macromolecular interactions on the kinetics of inactivation of trypsin and α-chymotrypsin

Johnson

Whateley

1981

Biochemical Journal

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“…This phenomenon has previously been observed and discussed (21,22) and suitable corrections were made. In this study all experimental data have been analyzed and the kinetic constants computed on the IBM 360165 computer using a slightly modified version of the program written by Hanson et 01.…”

Section: Methods Of Coiilputatiorzsupporting

confidence: 60%

pH Effects in trypsin catalysis

Kasserra¹,

Laidler²

1969

Can. J. Chem.

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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.

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“…The value of this rate could be determined either by measuring the rate of production of ninhydrin-positive materials in the absence of substrate, or by extrapolating a plot of vo against [So] to zero [So]. With oc-chymotrypsincatalysed reactions a satisfactory method of correcting for enzyme blank has been used by Ingles & Knowles (1966), which depends on the assumption that only the free enzyme (and not the Michaelis complex) can autolyse. In the present work a simpler procedure, that of subtracting the enzyme blank from each rate, was used, which does not greatly differ from the procedure of Ingles & Knowles (1966) unless [So] is larger than Km.…”

Section: Resultsmentioning

confidence: 99%

The pH-dependence of pepsin-catalysed reactions

Cornish‐Bowden

Knowles

1969

Biochemical Journal

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1. The pH-dependence of the pepsin-catalysed hydrolysis of three peptide substrates was studied by using a method for the continuous monitoring of the formation of ninhydrin-positive products. 2. Two peptide acid substrates, N-acetyl-l-phenylalanyl-l-phenylalanine and N-acetyl-l-phenylalanyl-l-phenylalanyl-glycine, show apparent pK(a) values of 1.1 and 3.5 in the plots of k(0)/K(m) versus pH. By contrast a neutral substrate, N-acetyl-l-phenylalanyl-l-phenylalanine amide, shows apparent pK(a) values of 1.0 and 4.7. 3. Together with the data of the preceding paper (Knowles, Sharp & Greenwell, 1969), these results are taken to indicate that the rate of pepsin-catalysed hydrolysis is controlled by the ionization of two groups, which on the free enzyme have apparent pK(a) values of 1.0 and 4.7. It is apparent that the anions of peptide acid substrates are not perceptibly bound to the enzyme, resulting in apparent pK(a) values of 3.5 for the dependence of k(0)/K(m) for these materials.

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The α-chymotryptic hydrolysis of glycine esters

Cited by 24 publications

References 17 publications

The effects of surface and macromolecular interactions on the kinetics of inactivation of trypsin and α-chymotrypsin

The effects of surface and macromolecular interactions on the kinetics of inactivation of trypsin and α-chymotrypsin

pH Effects in trypsin catalysis

The pH-dependence of pepsin-catalysed reactions

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