The first activation study of the β-carbonic anhydrases from the pathogenic bacteria Brucella suis and Francisella tularensis with amines and amino acids

Figures & data

Figure 1. Amino acids and amines 1–24 investigated as activators.

Table 1. Activation of hCA isozymes I, II and BsuCA 1/FtuCA, with L-Trp, at 25 °C, for the CO₂ hydration reaction²⁰.

Isozyme	kcat (s^−1)^a	KM (mM)^a	(kcat)L-Trp (s^−1)^b	KA (μM) L-Trp^c
hCA I^d	2.0 × 10^5	4.0	3.4 × 10^5	44
hCA II^d	1.4 × 10^6	9.3	4.9 × 10^6	27
BsuCA 1^e	6.4 × 10^5	16.4	25.9 × 10^5	1.25
FtuCA^e	9.8 × 10^5	11.0	15.7 × 10^5	34.1

^a Observed catalytic rate without activator. K_M values in the presence and the absence of activators were the same for the various CAs (data not shown).

^b Observed catalytic rate in the presence of 10 μM activator.

^c The activation constant (K_A) for each enzyme was obtained by fitting the observed catalytic enhancements as a function of the activator concentration^21–29. Mean from at least three determinations by a stopped-flow, CO₂ hydrase method²⁰. Standard errors were in the range of 5–10% of the reported values (data not shown).

^d Human recombinant isozymes, from Supuran⁸a.

^e Bacterial recombinant enzyme, this work.

Table 2. Activation constants of hCA I, hCA II and the bacterial β-CAs investigated here with amino acids and amines 1–24, by a stopped-flow CO₂hydrase assay²⁰.

No.	Compound	KA (μM)^a
		hCAI^b	hCAII^b	BsuCA1^c	FtuCA^c
1	L-His	0.03	10.9	1.76	40.7
2	D-His	0.09	43	12.3	78.3
3	L-Phe	0.07	0.013	1.16	69.1
4	D-Phe	86	0.035	1.21	75.0
5	L-DOPA	3.1	11.4	2.07	>100
6	D-DOPA	4.9	7.8	2.34	44.8
7	L-Trp	44	27	1.25	34.1
8	D-Trp	41	12	13.7	30.5
9	L-Tyr	0.02	0.011	1.38	>100
10	D-Tyr	0.04	0.013	0.95	>100
11	4-H2N-L-Phe	0.24	0.15	1.18	>100
12	Histamine	2.1	125	3.71	>100
13	Dopamine	13.5	9.20	1.54	>100
14	Serotonin	45	50	4.26	>100
15	2-Pyridyl-methylamine	26	34	1.62	46.3
16	2-(2-Aminoethyl)pyridine	13	15	5.20	>100
17	1-(2-Aminoethyl)-piperazine	7.4	2.30	43.1	51.8
18	4-(2-Aminoethyl)-morpholine	0.14	0.19	9.56	>100
19	L-Adrenaline	0.09	96	0.70	>100
20	L-Asn	11.3	>100	7.38	>100
21	L-Asp	5.20	>100	6.82	>100
22	L-Glu	6.43	>100	9.36	9.13
23	D-Glu	10.7	>100	1.39	>100
24	L-Gln	>100	>50	3.21	45.7

^a Mean from three determinations by a stopped-flow, CO₂ hydrase method²⁰. Standard errors were in the range of 5–10% of the reported values (data not shown).

^b Human recombinant isozymes, stopped flow CO₂ hydrase assay method²⁶.

^c Bacterial enzyme, this work.

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