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CAs catalyze the physiologically crucial reversible reaction of the carbon dioxide hydration to bicarbonate and protons. Herein, we investigated the sulfonamide inhibition profile of the recombinant β-CA (CynT2) identified in the genome of the Gram-negative bacterium, Escherichia coli. This biocatalyst is indispensable for the growth of the microbe at atmospheric pCO2. CynT2 was strongly inhibited by some substituted benzene-sulfonamides, and the clinically used inhibitor sulpiride (KIs in the range of 82–97 nM). This study may be relevant for identifying novel CA inhibitors, as well as for another essential part of the drug discovery pipeline, such as the structure-activity relationship for this class of enzyme inhibitors.
Table 1. Kinetic parameters for the CO2 hydration reaction catalyzed by the human α-CAs and bacterial CAs (α-, β-, γ- and ι-CAs).
Organism |
Acronym |
Class |
kcat (s-1) |
kcat/Km (M-1 x s-1) |
KI (acetazolamide) (nM)
|
Homo sapiens a |
hCA I |
α |
2.0 x 105 |
5.0 x 107 |
250 |
|
hCA II |
α |
1.4 x 106 |
1.5 x 108 |
12 |
Vibrio cholerae |
VchCAα |
α |
8.2 x 105 |
7.0 x 107 |
6.8 |
Escherichia coli |
CynT2 |
β |
5.3 x 105 |
4.1 x 107 |
227 |
Vibrio cholerae |
VchCAβ |
β |
3.3 x 105 |
4.1 x 107 |
451 |
Porphyromonas gingivalis |
PgiCAβ |
β |
2.8 x 105 |
1.5 × 107 |
214 |
Helicobacter pylori |
HpyCAβ |
β |
7.1 x 105 |
4.8 x 107 |
40 |
Porphyromonas gingivalis |
PgiCAγ |
γ |
4.1 x 105 |
5.4 × 107 |
324 |
Vibrio cholerae |
VchCAγ |
γ |
7.3 x 105 |
6.4 x 107 |
473 |
Burkholderia territorii |
BteCAι |
ι |
3.0 x 105 |
9.7 × 107 |
65 |
In Table 2 is reported the inhibition profile of CynT2. The comparative analysis was carried out analyzing the CynT2 inhibitory behavior with those obtained for the enzyme VchCAβ (β-CA form Vibrio cholerae) and the two human γ-CA isoforms, hCA I and hCA II.
Table 2. Inhibition of the human isoforms hCA I and hCA II and the two bacterial β-CAs (CynT2 and VchCAβ) with sulfonamides 1-24 and the clinically used drugs AAZ-EPA.
Inhibitor |
KI*(nM) hCA Ia hCA IIa CynT2 VchCAβa |
|||
1 |
28000 |
300 |
705 |
463 |
2 |
25000 |
240 |
790 |
447 |
3 |
79 |
8 |
457 |
785 |
4 |
78500 |
320 |
3015 |
>10,000 |
5 |
25000 |
170 |
2840 |
>10,000 |
6 |
21000 |
160 |
3321 |
>10,000 |
7 |
8300 |
60 |
>10000 |
>10,000 |
8 |
9800 |
110 |
>10000 |
9120 |
9 |
6500 |
40 |
2712 |
>10,000 |
10 |
7300 |
54 |
8561 |
>10,000 |
11 |
5800 |
63 |
6246 |
879 |
12 |
8400 |
75 |
4385 |
4450 |
13 |
8600 |
60 |
4122 |
68,1 |
14 |
9300 |
19 |
440 |
82,3 |
15 |
5500 |
80 |
6445 |
349 |
16 |
9500 |
94 |
2340 |
304 |
17 |
21000 |
125 |
502 |
3530 |
18 |
164 |
46 |
205 |
515 |
19 |
109 |
33 |
416 |
2218 |
20 |
6 |
2 |
726 |
859 |
21 |
69 |
11 |
473 |
4430 |
22 |
164 |
46 |
93 |
757 |
23 |
109 |
33 |
322 |
817 |
24 |
95 |
30 |
82 |
361 |
AAZ |
250 |
12 |
227 |
4512 |
MZA |
50 |
14 |
480 |
6260 |
EZA |
25 |
8 |
557 |
6450 |
DCP |
1200 |
38 |
>10000 |
2352 |
DZA |
50000 |
9 |
629 |
4728 |
BRZ |
45000 |
3 |
2048 |
845 |
BZA |
15 |
9 |
276 |
846 |
TPM |
250 |
10 |
3359 |
874 |
ZNS |
56 |
35 |
3189 |
8570 |
SLP |
1200 |
40 |
97 |
6245 |
IND |
31 |
15 |
2392 |
7700 |
VLX |
54000 |
43 |
2752 |
8200 |
CLX |
50000 |
21 |
1894 |
4165 |
SLT |
374 |
9 |
285 |
455 |
SAC |
18540 |
5959 |
6693 |
275 |
HCT |
328 |
290 |
5010 |
87 |
FAM |
922 |
58 |
2769 |
- |
EPA |
8262 |
917 |
2560 |
- |