Cobalt -carbonic anhydrase

As examples. Table 8 records some observations on d—d and charge transfer absorption bands in metal/protein systems. The examination of the spectrum of cobalt carbonic anhydrase (d—d) and of iron conalbumin (charge-transfer) permitted a prediction of the ligands from the protein to the metal. The predictions have now been substantiated by other methods. 

Given the reaction and the very high stability constants involved, the production of cobalt carbonic anhydrase would require a solution not of ACS-grade cobalt nitrate but a 99.999999999999. .. 999% pure cobalt nitrate solution. What happened in the lab synthesis was that trace metals in the ACS-grade salt were selectively bound to the apo-carbonic anhydrase because their stability constant advantage was orders of magnitude over that of cobalt. The sample used to discover this was sub-milligram in mass. 

The d-d, CD and MCD spectra of cobalt carbonic anhydrase are pH dependent, as noted earlier. In general the d-d and MCD spectra of the enzyme in acidic pH resemble those of Co11 complexes of tetrahedral geometry. The spectra at alkaline pH are different from the spectra at acid pH, and suggest the presence of a distorted five-coordinate cobalt(II) centre. Thus it appears that in the active form of the enzyme the metal is present in a five-coordinate geometry. 

The circular dichroic spectrum of cobalt alkaline phosphatase (Fig. 16) shows more clearly the complexity of the visible absorption. Although it can not be ruled out that the spectrum of this Co (I I) enzyme represents two slightly different Co(II) sites, there are striking similarities with Co(II) carbonic anhydrase, which has only one metal-binding site. At high pH, cobalt carbonic anhydrase and cobalt alkaline phosphatase have several spectral features in common, and both may have a similar kind of irregular coordination. It should be noted, however, that the absorption coefficient for Co(II) alkaline phosphatase per equivalent of activity-linked metal ion is only half of the value for Co(II) carbonic anhydrase. 

Cobalt has recently been used as an ESR active substitute in zinc metalloenzymes. Whilst liquid helium temperatures may be needed and theoretical aspects of the spectra are not yet as well understood, cobalt has two important advantages over copper as a metal substitute, namely that many cobalt derivatives show some enzymic activity (e.g. cobalt in carbonic anhydrase, alkaline phosphatase and superoxide dismutase) and that g values and hyperfine splitting are more sensitive to ligand environment, particularly when low spin. ESR data have been reported for cobalt substituted thermolysin, carboxypeptidase A, procarboxypeptidase A and alkaline phosphatase [51]. These are all high spin complexes. Cobalt carbonic anhydrase has been prepared and reacted with cyanide [52]. In... 

Nickel is required by plants when urea is the source of nitrogen (Price and Morel, 1991). Bicarbonate uptake by cells may be limited by Zn as HCOT transport involves the zinc metal-loenzyme carbonic anhydrase (Morel et al., 1994). Cadmium is not known to be required by organisms but because it can substitute for Zn in some metalloenzymes it can promote the growth of Zn-limited phytoplankton (Price and Morel, 1990). Cobalt can also substitute for Zn but less efficiently than Cd. 

Several other nmr procedures have been used for the determination of fractionation factors. These have advantages in some systems. Instead of determining the effect of the concentration of an exchanging site on the averaged chemical shift, the effect on the averaged relaxation rate of water protons can be used in a very similar way (Silverman, 1981 Kassebaum and Silverman, 1989), For example, addition of the enzyme Co(ii)-carbonic anhydrase to an aqueous solution increases the observed value of XjT because the proton-relaxation rate is the average of that for the bulk solvent (cfl. 0.3 s ) and that for water bound to the cobalt ca. 6x 10 s ). The average is different in an H2O/D2O mixture if the bulk solvent and the Cobound solvent have different deuterium contents, and it has been used to determine a value for the fractionation factor of Co-bound water molecules in the enzyme. 

As an example of tetra-coordinate cobalt(II) systems, the NMRD profile of cobalt(II)-substituted carbonic anhydrase (MW 30,000) at high pH is reported (Fig. 14). The metal ion is coordinated to three histidines and to a hydroxide ion (48). The NMRD profile shows a cos Cg dispersion centered around 10 MHz, which qualitatively sets the correlation time around 10 s. As the reorientational correlation time of the molecule is much longer, this value is a measure of the effective electronic relaxation time. A quantitative... 

Fig. 14. Paramagnetic enhancements to water NMRD profiles for solutions of cobalt(II) human carbonic anhydrase I at pH 9.9 and 298 K ( ) (48,49) and for solutions of the nitrate adduct of cobalt(II) bovine carbonic anhydrase II at pH 6.0 and 298 K ( ) (126). The dashed line shows the best fit profile of the former data calculated with including the effect of ZFS, whereas the dotted line shows the best fit profile calculated without including the effect of ZFS.

The cobalt-substituted carbonic anhydrase has been extensively studied as it offers easily measurable pH-dependent electronic spectra... 

Hydroxide ion coordinated to cobalt(IIl) has been observed to function as a nucleophile in both intermolecular and intramolecular reactions. The possibility that coordinated hydroxide is directly involved in the catalytic action of some metalloenzymes such as carbonic anhydrase has prompted a number of investigations of metal hydroxide reactivity towards organic substrates. Much of this chemistry has been reported and reviewed.21,23... 

Carbonic anhydrase is a zinc(II) metalloenzyme which catalyzes the hydration and dehydration of carbon dioxide, C02+H20 H+ + HC03. 25 As a result there has been considerable interest in the metal ion-promoted hydration of carbonyl substrates as potential model systems for the enzyme. For example, Pocker and Meany519 studied the reversible hydration of 2- and 4-pyridinecarbaldehyde by carbonic anhydrase, zinc(II), cobalt(II), H20 and OH. The catalytic efficiency of bovine carbonic anhydrase is ca. 108 times greater than that of water for hydration of both 2- and 4-pyridinecarbaldehydes. Zinc(II) and cobalt(II) are ca. 107 times more effective than water for the hydration of 2-pyridinecarbaldehyde, but are much less effective with 4-pyridinecarbaldehyde. Presumably in the case of 2-pyridinecarbaldehyde complexes of type (166) are formed in solution. Polarization of the carbonyl group by the metal ion assists nucleophilic attack by water or hydroxide ion. Further studies of this reaction have been made,520,521 but the mechanistic details of the catalysis are unclear. Metal-bound nucleophiles (M—OH or M—OH2) could, for example, be involved in the catalysis. 

Most work has involved the cobalt(II) carbonic anhydrase, and some illustrations will be given. Work with metallocarbonic anhydrases has been reviewed.482... 

The purpose of the present review is to summarize how cobalt-linked absorption spectra and other physical properties have been utilized in attempts to elucidate relations between structure and function in these enzymes. The emphasis will be on carbonic anhydrase not only because it reflects the author s own interests, but mainly because it is the most extensively studied cobalt enzyme. Its environmentally-sensitive absorption spectrum has furnished essential information as to the role of the metal ion in the catalytic reaction. For other enzymes, the probe properties of cobalt are just beginning to be explored, but significant advances have recently been reported (7). 

Carboxypeptidase A was the first metalloenzyme where the functional requirement of zinc was clearly demonstrated (9, 92). In similarity to carbonic anhydrase, the chelating site can combine with a variety of metal ions (93), but the activation specificity is broader. Some metal ions, Pb2+, Cd2+ and Hg2+, yield only esterase activity but fail to restore the peptidase activity. Of a variety of cations tested, only Cu2+ gives a completely inactive enzyme. In the standard peptidase assay, cobalt carboxypeptidase is the most active metal derivative, while it has about the same esterase activity as the native enzyme ((93, 94), Table 6). Kinetically, the Co(II) enzyme shows the same qualitative features as the native enzyme (95), and the quantitative differences are not restricted to a single kinetic parameter. 

In the case of cobalt substituted Zn-fingers [102], the differences between the chemical shifts for corresponding resonances in the Co(II) and Zn(II) complexes allow the determination of the orientation and anisotropy of the magnetic susceptibility tensor [103]. Similar studies are available for pseudotetrahedral Co(II) in the zinc site of superoxide dismutase [104] and five coordinated carbonic anhydrase derivatives [105]. 

Fig. 9.5.60 MHz H NMR Modeft spectra of cobalt-substituted carbonic anhydrase (MW 30,000) adducts with iodide and oxalate. The 7) values for some signals obtained with Eq. (9.1) (see later) are indicated. The dashed signals disappear in D20 [20].

Taylor, P. W., Feeney, J., and Burgen, A. S. V. Investigation of the mechanism of ligand binding with cobalt (II) human carbonic anhydrase by lH and 19F nuclear magnetic resonance spectroscopy. Biochemistry JO, 3866-3875 (1971). 

As with carbonic anhydrase the metal is in a cleft that exposes the active site. Interestingly the metal-free enzyme is inactive but the cobalt and nickel analogues are more active. It appears that the transition state complex, where the terminal amino acid side chain is held in place while the peptide bond is hydrolysed, requires six-fold co-ordination. The activation energy required to change from the tetrahedral to octahedral geometries is higher for zinc than the other metals. 

Metals are used not only as labels in histochemistry and immunochemistry, but also for studying protein structure and properties. Some metal ions can serve as valuable probes by replacing the original ions in different metaloenzymes or other metaloproteins. For instance, cobalt can replace zinc on the active side of carboxy-peptidase, aldolase, carbonic anhydrase, phosphatase or yeast alcohol dehydrogena. ... 

Another way of bringing reactants into close proximity, which is encountered commonly in transition metal chemistry, is through metal ion complexation. The coordination of a reactant to a metal ion complex often activates its reactivity and can bring the reactant into close proximity with a second reactant or with a catalytic group. One example, shown in Fig. 6, is a zinc (11) complex of 1,5,9-triazacyclononane, as a model for the enzyme carbonic anhydrase, which contains a zinc (11) cofactor in its active site (4). In the aqua complex, the bound water molecule has a dramatically reduced pKa value of 7.3, which is similar to the pKa of the active site nucleophihc water. The corresponding cobalt (111) complex catalyzed ester hydrolysis at twice the rate because Co(lll) can coordinate both the hydroxide nucleophile and the ester carbonyl via a... 

Figure 6 Zinc (II) and cobalt (III) complexes of 1, S,9-triazacyclododecane as mimics of carbonic anhydrase.

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