Carbonic anhydrase inhibitors. Cloning, characterization and inhibition studies of the cytosolic isozyme III with anions

Figures & data

Figure 1.  Alignment of the amino acid sequence of isoform CA III with that of isozymes CA I and II (CA I numbering system used). Thirty-six active site residues previously defined as forming the active site [27] are indicated by a mixture of asterisk, “plus” and “z” signs above the CA I sequence. Seventeen residues known to participate in a network of hydrogen bonds and being involved in the binding of inhibitors/activators [28] are indicated by “plus” and “z” above the sequence; the latter sign indicates the three zinc-liganded histidine residues (His94, 96 and 119). Conserved amino acids in the three isoforms are indicated by a closed box.

Table I.  Kinetic parameters for the CO₂ hydration reaction catalysed by the recombinant cytosolic hCA isozymes I-III, at 20°C and pH 7.5, and their inhibition data with acetazolamide AAZ(5-acetamido-1,3,4-thiadiazole-2-sulfonamide), a clinically used drug [1].

Isozyme	Activity level	kcat (s^− 1)	kcat/Km (M^− 1 s^− 1)	KI (acetazolamide) (nM)
hCA I	moderate	2.0 × 10^5	5.0 × 10^7	250
hCA II	very high	1.4 × 10^6	1.5 × 10^8	12
hCA III	very low	1.3 × 10^4	2.5 × 10^5	2 × 10^5

Figure 2.  SDS PAGE for the hCA III–GST fusion protein. The band of the fusion protein (with a molecular weight of around 50 kDa) appears only after addition of IPTG to the growth medium.

Figure 3.  SDS PAGE for the hCA I- III proteins. Lanes: 1 = Ladder; 2 = hCA I; 3 = hCA II; 4 = hCA III. hCA I and II were from Sigma-Aldrich, whereas hCA III is the recombinant protein prepared by the GST fusion method, after the final purification steps.

Table II.  Inhibition of recombinant isozymes hCA I, II and III with anions by a stopped-flow kinetic assay monitoring the CO₂ hydration reaction, at 20°C and pH 7.5 [26].

	KI [mM]^#
Anion inhibitor	hCA I^a,b	hCA II^a,b	hCA III^a
F^−	>300	>300	78.5
Cl^−	6	200	0.98
Br^−	4	63	0.96
I^−	0.3	26	0.90
CN^−	0.0005	0.02	0.06
CNO^−	0.0007	0.03	0.57
SCN^−	0.2	1.6	0.09
N3^−	0.0012	1.5	0.08
HCO3^−	12	85	0.74
CO3^2 −	15	73	0.010
HSO3^−	18	89	1.06
NO3^−	7	35	117
NO2^−	8.4	63	53.2
HS^−	0.0006	0.04	0.08
SO4^2 −	63	>200	1.00
H2NSO3H^*	0.021	0.39	31.1
H2NSO2NH2	0.31	1.13	1.09
PhB(OH)2	58.6	23.1	21.3
PhAsO3H2^*	31.7	49.2	30.8

^# Errors were in the range of 3–5% of the reported values, from three different assays ^a Human recombinant isozymes ^b Data from refs. [32,33] * As sodium salt.

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