Hydrolysis of the dipeptide derivative 5 catalysed by carboxypeptidase A.

110

buffer might interfere with the inclusion of the substrate 5 within a cyclodextrin, so it was not used in these studies. A phosphate buffer was chosen instead as, despite having a minor inhibitory effect on carboxypeptidase A ,17 it was expected that it would not interfere with complexation o f the substrate

5

with a cyclodextrin. A pH of 7.5 was used, chosen as that where the enzyme shows maximum activity.17' 19 A previous report17 indicates that carboxypeptidase A activity increases as the ionic strength of the reaction mixture increases, up to a value of 0.25 mol dm*3. Above this, the activity of the enzyme is independent of ionic strength. In these studies, potassium chloride was added to each of the reaction mixtures to give an ionic strength of 0.5 mol dm '3, ensuring that the change in the ionic strength as the substrate is hydrolysed does not affect enzyme activity.

R eactions involving the hydrolysis o f the dipeptide derivative

5

by carboxypeptidase A were carried out at 298 K. They were monitored by acidifying aliquots with dilute hydrochloric acid, which denatured the enzyme and caused the substrate 5 and the A-protected glycine

109

to precipitate. The protonated forms of the

Results and Discussion, Chapter VII

substrate 5 and the product 109 are soluble in organic solvents, so they were separated from the other components of the reaction mixtures through extraction of the suspensions with ethyl acetate. Through analysis of these organic extracts using HPLC, the relative amounts of the substrate 5 and the product 109, and hence the extent of reaction at a given time, were determined. From this information, the rate o f reaction (v) of the substrate 5 was calculated.

Initially, the hydrolysis of the dipeptide derivative 5 by carboxypeptidase A (ca. 7.5 units dm*3) was examined in the absence of a cyclodextrin at concentrations of the substrate 5 over the range 0.001 - 0.30 mol dm '3 to study the effect on enzyme activity. As shown in Figure 22, the rate of hydrolysis of the peptide derivative 5 increases with increasing substrate 5 concentration up to a value of ca. 0.06 mol d n r3. Above this, the rate of reaction decreases with increasing substrate 5 concentration to approximately two-thirds of the maximum observed. These results show that the rate of hydrolysis decreases at high concentrations of the substrate 5, as is consistent with substrate inhibition of carboxypeptidase A. This substrate inhibition is further illustrated in Figure 23, which is a plot of the form derived by Lineweaver and B urk,113 with the reciprocal of the substrate 5 concentration used as the ordinate axis and the reciprocal of the rate of reaction used as the coordinate axis. The notable deviation from linearity of the plot near the coordinate axis is consistent with substrate inhibition.176

1.60E-06- 1.20E-06- 'c/> oo E 8.G O E R ­ 'S £ 4.00E-07- O.OOE+OO-

0.00E+00 8.00E-02 1.60E-01 2.40E-01 3.20E-01

[(S)-carbobenzoxyglycylphenylalanine (5)] (mol dm'3)

Figure 22. Rate of reaction plotted against substrate concentration for the hydrolysis of carbobenzoxyglycylphenylalanine

(5)

by carboxypeptidase A (ca. 7.5 units d n r3) in 0.05 mol d n r 3 pH 7.5 phosphate buffer (I = 0.5 mol d n r3) at 298 K.

1.80E+06 1.50E+06 co 1.20E+06

E

~o T_ 9.00E+05

o

£

* 6.00E+05 1— 3.00E+05 O.OOE+OO

O.OOE+OO 3.00E+01 6.00E+01 9.00E+01 1.20E+02

1/[(S)-carbobenzoxyglycylphenylalanine (5)] (mol'1 dm3)

Figure 23. Lineweaver-Burk plot for the hydrolysis of the peptide derivative 5 by carboxypeptidase A (ca . 7.5 units d n r3) in 0.05 mol dm '3 pH 7.5 phosphate buffer (I = 0.5 mol dm-3) at 298 K.

At concentrations of the substrate 5 greater than ca. 0.06 mol dm -3 substrate inhibition of carboxypeptidase A is observed. The use of ß-cyclodextrin (8) to limit substrate inhibition is impractical in this case as, due to limited solubility, it can be present in aqueous solution at a maximum concentration of ca. 0.016 mol dm-3.97 Therefore, the maximum reduction in substrate 5 concentration on addition of the cyclodextrin 8 is 0.016 mol dm*3. This would clearly have a negligible effect on substrate inhibition of carboxypeptidase A in the hydrolysis of the peptide 5.

Hydroxypropyl-ß-cyclodextrin (108), as used in the work discussed in Chapter VI of the Results and Discussion, has an increased aqueous solubility when compared to ß-cyclodextrin (8).174,175 The protonated form of 6A-amino-6A-deoxy-a-cyclodextrin (111) has a pA^a of 8.7,111 so the amine 111 would be present in solution predominantly in the protonated form at pH 7.5. This form o f the amine 111 also has an increased aqueous solubility when compared to ß-cyclodextrin (8).111 Thus, both the amine 111, available in the laboratory,120 and the modified cyclodextrin 108 can be present in solution at a greater concentration than ß-cyclodextrin (8), so the maximum amount of a guest they can remove from solution is larger. Since complexation of a guest by a host is a bimolecular reaction, a higher concentration of the host results in a greater proportion of the guest being complexed. Where the guest is an inhibitor of an enzyme, it was expected that a larger amount of the host would lead to less of the inhibitor free in solution and

Results and Discussion, Chapter VII

hence a greater effect of the host to limit inhibition. Thus, it was anticipated that the modified cyclodextrins 108 and 111 would be more effective than ß-cyclodextrin (8) to limit substrate inhibition of carboxypeptidase A through removing a greater proportion of the substrate 5 from solution.

The effect of each of the cyclodextrins 108 and 111 on substrate inhibition of carboxypeptidase A in the hydrolysis of the dipeptide derivative 5 was examined by repeating the previous experiments in the presence of the modified cyclodextrin 108 (0.186 mol dm-3) and the amine 111 (0.221 mol dm*3). Since the amine 111 is present mainly in the protonated form at pH 7.5, the ionic strength of the reaction mixtures containing the amine 111 was ca. 0.9 mol dm*3, compared to 0.5 mol dm*3 in the other cases. Based on previous studies,17 it is reasonable to assume that this change will have

a negligible effect on carboxypeptidase A activity.

The results of the experiments m onitoring the hydrolysis of the dipeptide derivative 5 by carboxypeptidase A in the presence of the cyclodextrins 108 and 111 are illustrated in Figure 24 with comparison to the case with no cyclodextrin present. They indicate that carboxypeptidase A retains activity in the presence of the modified cyclodextrins 108 and 111. At the lower concentrations o f the substrate 5 used, addition of each of the modified cyclodextrins 108 and 111 reduces the rate of reaction, markedly in the case of the amine 111. At the higher concentrations of the peptide derivative 5 used, substrate inhibition of carboxypeptidase A is observed in each case though the rate of reaction is greater in the presence of the cyclodextrins 108 and 111 than in the absence of a cyclodextrin. Further, the concentration of the substrate 5 at which the maximum rate of reaction occurs increases on the addition of each of the modified cyclodextrins 108 and 111.

These results are consistent with the cyclodextrins 108 and 111 complexing the substrate 5 and reducing its concentration free in solution. At the lower concentrations of the substrate 5 used, this results in less of the Michaelis complex being formed and hence a decrease in the observed rate of hydrolysis. At the higher concentrations of the

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4.00E -07 O.OOE+OO

0.00E+00 8.00E-02 1.60E-01 2.40E-01 3.20E-01

[(S)-carbobenzoxyglycylphenylalanine (5)] (mol dm'3)

Figure 24.

Rate of reaction plotted against substrate concentration for the hydrolysis of GS)-carbobenzoxyglycylphenylalanine (5) by carboxypeptidase A (ca. 7.5 units dm’3) in 0.05 mol dm-3 pH 7.5 phosphate buffer at 298 K in the presence of either no cyclodextrin (I = 0.5 mol dm-3) ( ♦ ) , hydroxypropyl-ß-cyclodextrin

(108)

(0.186 mol dm-3, I = 0.5 mol dm-3) (0) or the amine (111) (0.221 mol d n r3, 1 = 0.9 mol d n r 3) ( • ) .

substrate 5 used, decreasing its concentration free in solution results in an increase in the proportion of the Michaelis complex present in solution and hence an increase in the rate of reaction o f the substrate 5. That is, the cyclodextrins

108

and

111

limit substrate inhibition and increase the concentration at which the maximum rate of reaction occurs in the carboxypeptidase A catalysed hydrolysis of the peptide derivative 5, through sequestering the substrate 5 and reducing its concentration free in solution.

It was anticipated that cyclodextrins would have a greater effect on substrate inhibition of an enzyme which occurs at lower concentrations of substrate than is the case for the hydrolysis of the peptide derivative 5 by carboxypeptidase A. In such a system, it was expected that less substrate would have to be rem oved from solution by a cyclodextrin for it to have a significant effect on enzyme activity.

Experiments reported by Bunting22' 26,177,178 and others18' 21 on the hydrolysis of (S)-lactate derivatives of A-benzoylglycine by carboxypeptidase A have shown that substrate inhibition of the enzyme occurs at much lower substrate concentrations than observed for the hydrolysis of the peptide derivative 5. W hile dependent on the side chain of the lactate portion of the molecule, the maximum rates of reaction were observed at substrate concentrations of ca. lO 3 mol dm 3, with only negligible activity being

Results and Discussion, Chapter VII

observed above 10 x 10'3 mol d m 3. Thus, it was anticipated that the hydrolysis of an A-benzoylglycyllactate by carboxypeptidase A would be more suitable to examine the effect of cyclodextrins on substrate inhibition.

The lactate chosen for study was (.S)-A-benzoylglycyl-ß-phenyllactate (6), which is hydrolysed by carboxypeptidase A as shown in Scheme 31. It was anticipated that a cyclodextrin would complex the lactate 6 reducing its concentration free in solution, hence limiting substrate inhibition of carboxypeptidase A.

Scheme 31. Hydrolysis of the lactate derivative 6 catalysed by carboxypeptidase A.

In document Cyclodextrins in conjunction with and as alternatives to enzymes (Page 108-113)