Activation of the β-carbonic anhydrase from the protozoan pathogen Trichomonas vaginalis with amines and amino acids

Figures & data

Figure 1. Amino acids and amines 1–24 investigated as CAAs against TvaCA1. Some β-CAs have a so-called closed active site (at pH < 8.3), in which the fourth zinc ligand is an aspartate residue, and thus these enzymes are devoid of CO₂ hydrase activity^21–23. However, at pH values > 8.3, the aspartate is involved in a hydrogen bond with an adjacent Arg residue, and an incoming water molecule/hydroxide ion replaces the aspartate as a zinc ligand, providing an open active site and thus a catalytically active enzyme^21–23.

Table 1. Activation of hCA isozymes I, II, EcoCAβ and TvaCA1 with L-Trp, measured at 25 °C¹⁵.

Isozyme	class	kcat* (s^−1)	KM* (mM)	(kcat)L-Trp** (s^−1)	KA*** (µM) L-Trp
hCA I^a	α	2.0 × 10^5	4.0	3.4 × 10^5	44.0
hCA II^a	α	1.4 × 10^6	9.3	4.9 × 10^6	27.0
EcoCAβ^b	β	5.3 × 10^5	12.9	1.8 × 10^6	18.3
TvaCA1^c	β	4.9 × 10^5	6.1	3.0 × 10^6	5.1

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

**Observed catalytic rate in the presence of 10 µM activator.

***The activation constant (K_A) for each enzyme was obtained by fitting the observed catalytic enhancements as a function of the activator concentration¹⁵.

^aHuman recombinant isozymes, from ref.¹¹. ^bBacterial recombinant enzyme, from ref.^14b. ^cThis work, protozoan enzyme. All values are mean from at least three determinations by the stopped-flow, CO₂ hydrase method¹⁵. Errors were in the range of 5–10% of the reported values (data not shown).

Table 2. Activation constants of hCA I, hCA II and the bacterial enzyme EcoCAβ (E. coli) and the protozoan TvaCA1 (T. vaginalis) with amino acids and amines 1–24, by a stopped-flow CO₂ hydrase assay¹⁵.

		KA (µM)*
No.	Compound	hCA I^a	hCA II^a	EcoCAβ^b	TvaCA1^c
1	L-His	0.03	10.9	36.0	20.1
2	D-His	0.09	43	23.7	24.5
3	L-Phe	0.07	0.013	12.0	23.6
4	D-Phe	86	0.035	15.4	16.3
5	L-DOPA	3.1	11.4	10.7	12.1
6	D-DOPA	4.9	7.8	3.14	11.0
7	L-Trp	44	27	18.3	5.1
8	D-Trp	41	12	11.5	3.6
9	L-Tyr	0.02	0.011	9.86	4.9
10	D-Tyr	0.04	0.013	17.9	3.0
11	4-H2N-L-Phe	0.24	0.15	7.34	3.5
12	Histamine	2.1	125	18.5	8.4
13	Dopamine	13.5	9.2	11.3	12.6
14	Serotonin	45	50	2.76	9.1
15	2-Pyridyl-methylamine	26	34	48.7	9.5
16	2-(2-Aminoethyl)pyridine	13	15	17.2	12.0
17	1-(2-Aminoethyl)-piperazine	7.4	2.3	14.1	11.8
18	4-(2-Aminoethyl)-morpholine 0.14	0.19	17.4	14.5
19	L-Adrenaline	0.09	96.0	9.15	8.3
20	L-Asn	11.3	>100	49.5	32.5
21	L-Asp	5.20	>100	18.9	>50
22	L-Glu	6.43	>100	18.0	>50
23	D-Glu	10.7	>100	11.4	>50
24	L-Gln	>100	>50	49.2	26.9

*Mean from three determinations by a stopped-flow, CO₂ hydrase method¹⁵. Errors were in the range of 5–10% of the reported values (data not shown).

^a Human recombinant isozymes, from ref.^8a.

^b Bacterial recombinant enzyme, ref.^14b.

^c Protozoan recombinant enzyme, this work.

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