Human carbonic anhydrase B

The buried Cys-212 of human carbonic anhydrase B (3 pM) is virtually unreactive towards 2-chloromercuric-4-nitrophenol (60 pM) at pH 9.2, but upon the addition of only 40 pM CN , the half-life drops to 10 minutes which is an, at least, 75-fold rate enhancement. On first analysis, this would suggest that inhibitor binding to the enzyme has produced a conformational change or altered the — SH environment of the Cys—212. This is unexpected. How would you prove by kinetic experiments that the CN is binding to the mercury compound and not the enzyme and that this is changing the reactivity. The rate reaches a constant value at high [CN ]. 

Figure B3.1.3 An isoelectric focusing (IEF) gel, pH 3 to 10. Lane 1, 4 pg purified egg white cystatin. Lane M, broad-range pi standards trypsinogen (pi 9.3), lentil lectin-basic band (pi 8.65), lentil lectin-middle band (pi 8.45), lentil lectin-acidic band (pi 8.15), myoglobin-basic band (pi 7.35 visible as a broad band), myoglobin-acidic band (pi 6.85), human carbonic anhydrase B (pi 6.55), bovine carbonic anhydrase (pi 5.85), a-lactoglobulin A (pi 5.20), soybean trypsin inhibitor (pi 4.55), and amyloglucosidase (pi 3.50) in order shown from top of gel. The pi values of the two purified egg white cystatin isomers were determined to be 6.6 (upper band) and 5.8 (lower band). Adapted from Akpinar (1998) with permission from author.

The structure of the human carbonic anhydrase B-imidazole complex involves five-coordinate zinc, the aqua group being retained.486 This is an important result in view of the competitive inhibition by imidazole. Sulfonamides displace the aqua ligand, being bound to the zinc by an O or N donor atom, with a second O atom forming an additional long bond to the zinc, making it... 

It should be noted that the kinetics for the human B isoenzyme are more complicated in that the pH dependence indicates that additional groups influence the rate. Studies with the isoenzyme carboxymethylated at His-200 prepared from 13C-labelled bromoacetate show that the pH dependence of the 13C NMR signal can be fitted to a curve with two pKa values of 6.0 and 9.2, but not to a curve with a single pKa. The second group could be the imidazole side-chain of His-200. Paramagnetically shifted 13C NMR resonances in the modified Co11 human carbonic anhydrase-B have been located by a novel method 498 This should allow the confirmation of an earlier postulate that the carboxymethyl carboxylate is a ligand for zinc in the modified enzyme. 

The presence of imidazole groups in the active site region of human carbonic anhydrase B has, in fact, been demonstrated by chemical modification. Thus, bromoacetate reacts specifically with the 3 -N of a histidine residue to give a partially active monocarboxymethyl enzyme (65). The reaction depends on the initial combination of the bromoacetate ion with the anion binding site (65,83). In a detailed study, Bradbury (83) has shown that the irreversible reaction at saturation with iodoacetate... 

Fig. 13. The pH dependence of the esterase activity of native and carboxymethyl-ated human carbonic anhydrase B. The term CM-enzymes refers to Zn(II)-enzyme ( . A) and Co(II)-enzyme (O, A) which had been carboxymethylated with bromo-acetate. Relative activity was estimated in each experiment by taking 100% as the value corresponding to maximal activity of the same enzyme at high pH. This value was 17 min-1 for the unreacted enzyme and 3.5 and 2.1 min-1 for the Zn(II)-and Co(II)-carboxymethylated enzymes, respectively. From Whitney et al. (65)...

A second histidine residue can be modified at the 3 -position in human carbonic anhydrase B with a N-chloroacetyl sulfonamide (84, 85) yielding an inactive product. 

Table 7. Apparent stability constants for metal binding in procarboxypeptidase A, carboxypeptidase A and human carbonic anhydrase B...

The enzyme also catalyses the hydrolysis of various esters such as 4-nitrophenyl acetate and sultones. A number of slightly different enzymes, carbonic anhydrases A, B and C occur in different organisms. The most well characterised enzymes are the bovine and human carbonic anhydrases B, which are monomeric and contain one tightly bound zinc per 30000 molecular weight. 

Figure 4. ESR spectra of Cu(II) human carbonic anhydrase B plus or (A) and...

Fig. 3.1. Reaction of human carbonic anhydrase B with tetranitromethane. The reaction mixture contained 5 ml of 0.3 % protein and 25 /il of tetranitromethane, buffered at pH 7.1 with O.I M sodium phosphate, or at pH 8.4 and 10.2 with 0.1 M Tris-H2S04. (Data from Dorner 1971).

Materials. Electrophoretically purified human carbonic anhydrase B (hCAB), also referred to as human carbonic anhydrase I, was purchased from the Sigma Chemical Co. (lot 104F93201). Electrophoresis grade urea was purchased from Fisher Scientific. Human carbonic anhydrase B stock solutions were stored at 4 C and used within four weeks, while urea solutions were prepared fresh prior to each experiment... 

Based on these findings, extinction coefficients for the various conformational species were measured under the following conditions OM urea for native hCAB, 8M urea for the denatured conformer, incubation in 5M urea for 90-120 minutes to estimate the extinction coefficient for X, and incubation in SM urea for an extended period of time (2 3 weeks was used in this study) to determine the extinction coefficient for conformer I. Initial extinction coefficients evaluations at 238 nm and 292 nm for the various human carbonic anhydrase B conformers were determined in O.IM Tris-HCl (pH=7.45) at 23 C. The results are presented in Table 2. It is evident that while there is a substantial (-25-35%) difference in extinction coefficients between the native and denatured states, the intermediate conformations X and I could not be separated spectroscopically, and therefore, UV absorbance cannot be used to determine the concentration of species X and I separately. Instead, the overall concentration of intermediate species, Z where [Z]=[X]+[q, was monitored over the course of a refolding experiment... 

The hydration of CO2 occurs at a relatively slow rate in the absence of a catalyst, with a rate constant of 0.037 s at 25 °C (equation 12). The enzyme catalyzes the reaction by a factor of lO at pH 7, the human isoenzyme C being more active than human carbonic anhydrase B by a factor of 30. Bovine B enzyme is also of high activity. The value for binding CO2 is nearly independent... 

Nome et al. [4Z1] recently reported on the competition between Cl" and the non-coordinative anion Au(CN)2 for binding to human carbonic anhydrase B and C and this observation suggests that the halide ion binding may be of a non-coordinative type. 

In view of the potential role of this exchange affecting the chemical shift, it is important to conduct the appropriate experiments (varying temperature, pH, solvent counterions, etc.) to rule in or out the presence of chemical exchange whenever the shift is being used to report on the nature of the protein ligands. Specific examples are the shift of the A-site Cd in alkaline phosphatase (AP) from 122 ppm in Tris-acetate to 169 ppm in Tris-chloride and in human carbonic anhydrase B (HCAB) where the replacement of the Cd-bound H2O or OH with Cl results in a shift from 145 ppm to 240 ppm. Both of these examples illustrate the deshielding affect of the metal bound Cl. ... 

The binding of 2-acetamido-5-N-(bromoacetyl)sulphonamido-1,3,4-thiadiazole ( N-Bromoacetyl-Acetazolamide ) at histidine sites in human carbonic anhydrase B has been studied by the use of the acetyl- C-labelled drug. The results suggest that the histidine sites reacting with this compound are possibly also concerned in the catalytic action of the enzyme. ... 

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