Question

The enzyme carboxypeptidase catalyses the hydrolysis of polypeptides and here we consider its inhibition. The following results were obtained when the rate of the enzymolysis of carbobenzoxy-glycyl-D-phenylalanine (CBGP) was monitored without inhibitor, where All rates in this problem were measured with the same concentration of enzyme and are relative to the rate measured when [CBGP]_o = 0.05 mol dm^-3 in the absence of inhibitor. When l.0 x 10^-3 mol.dm^-3 phenylbutyrate ion was added to a solution containing the enzyme and substrate, the following results were obtained: In a separate experiment, the effect of 5.0 x 10^-2 mol.dm^-3 benzoate ion was monitored and the results were: Determine the K_m, v_max, turnover frequency, catalytic efficiency, and the mode (mechanism) of inhibition of carboxypeptidase by the phenylbutyrate ion and benzoate ion.

Answer 1

Looking to the first table, you have to analyse if the reaction rate reference (i.e., for [CBGP]o = 0.05 mol/L) is correct. At [CBGP]o = 5.81x10-2 M (higher conc.) the relative reaction rate is 0.859 (lower). This situation is not acceptable. Thus I will correct the reference value to a more plausible value [S] = [CBGP]o = 0.10 mol/L.

To find Km, plot reaction rate vs. [S].

[S]	r. rate
0	0
0.0125	0.398
0.0384	0.669
0.0581	0.859
0.0913	1
0.1	1

[S]	Inhib PhBu
0	0
0.0125	0.172
0.0250	0.301
0.0400	0.344
0.0550	0.548

[S]	Inhib Benz
0	0
0.0175	0.183
0.0250	0.201
0.0500	0.231
0.1000	0.246

Something is also wrong with the experimental data (see graph “inhib PhBut”)

Read on the “no inhibitor” graph:

Km is [S] for v=vmax/2 . As relative value vmax = 1 For v=0.5 [S] = Km = 0.022

Catalytic efficiency kcat = vmax/[carboxypeptidase]o,      no data to calculate it

For the mechanism of inhibition you have to use the Lineweaver-Burk plot.

But you don’t have all necessary and good experimental data. Verify them first.