Cobalt -substituted carbonic anhydrase

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

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].

It is convenient to discuss the cobalt-substituted carbonic anhydrase enzyme, since its electronic spectra are markedly pH-dependent and easy to measure (Figures 2.7 and 2.8). The spectra are well-shaped, and a sharp absorption at 640 nm is present at high pH and absent at low pH. Whereas CoHCA I is almost entirely in the low-pH form at pH 5.7, this is not true for the CoBCA II isoenzyme. The acid-base equilibrium for Co-substituted carbonic anhydrase (deprotonation of the metal-coordinated water) involves three species ... 

Electronic spectra of Co(TPyMA)OH2 (Table 2.3) at various pH values. Note the similarity to the electronic spectra of cobalt-substituted carbonic anhydrase at various pH values as reported in Figure 2.1... 

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... 

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... 

Values of microconstants associated with acid-base equilibria in cobalt(ll)-substituted carbonic anhydrases. ... 

Types of pH dependences observed for the affinity constants of inhibitors for cobalt(II)-substituted carbonic anhydrases. p fa[E] represents the main pKa value of the enzyme, p/f [I] that of the inhibitor, if present. ... 

A study of the anion inhibition of Cobalt(II) HCAC CO2 hydration activity reported a loss of inhibitory power of anions at high pH(21). Further scrutiny of the high pH anion inhibition of metal substituted carbonic anhydrase is certainly in order. If, as it appears, high pH anion activity inhibition is a property confined to the natural substrate of the Zinc(II) enzyme, the implications for conclusions drawn from studies of model substrates and metal substituted enzymes need to be carefully scrutinized. 

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. 

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]. 

It appears that cobalt plays a particularly important role in the growth of cyanobacteria (Saito et al, 2002 Sunda and Huntsman, 1995b). Both Prochlorococcus and Synechococcus show an absolute cobalt requirement that zinc cannot substitute for (Figure 18(a)). The growth rate of Synechococcus is little affected by low zinc concentrations, except in the presence of cadmium which then becomes extremely toxic (Saito et al, personal communication). The biochemical processes responsible for the major cellular utilization of zinc and cobalt in marine cyanobacteria are unknown, however. These metals may be involved in carbonic anhydrase and/or other hydrolytic enzymes. Cobalamin (vitamin B12) synthesis is a function of cobalt in these organisms, yet B12 quotas tend to be very small (on the order of only 0.01 p.mol (mol C) ) and hence are not likely represent a significant portion of the cellular cobalt (Wilhelm and Trick, 1995). 

What are the essential chemical properties provided by zinc and its ligands in carbonic anhydrase (It may be noted that replacement of zinc by cobalt and cadmium has been carried out and for the cobalt substitution there is no change in properties while... 

Electronic spectra of cyanide (— and thiocyanate (- -) adducts of cobalt(II)-substituted bovine carbonic anhydrase II. 

The inhibition of carbonic anhydrase by anions has long been recognized. The inhibitory effect of Cl was noted by early investi-gators(19). A later study of anion inhibition by stopped-flow techniques was compromised by the presence of 80mM Cl in buffers used (20). The inhibition of the hydrase activity of the Cobalt(II) substituted enzyme has been investigated over the full pH range(21). Anionic inhibition of esterase activity has been studied by initial rate techniques(11-13) and by complexometric titration(22). None of the work thus far published has included full scale Michaelis-Menten analysis of the inhibition of the native Zinc(II) enzyme towards its natural substrate over an extended pH range. 

In an attempt to decide between these two possibilities, Lanir et al. have investigated the relaxation rates of the exchangeable water molecules in the inner coordination sphere of the metal in manganese-substituted bovine carbonic anhydrase B. Their conclusion is that one water molecule (and not a hydroxide ion) is directly bound to the metal at high pH values while below the pKa there is no rapidly exchanging water bound to the metal ion (cf. the conclusions of Koenig and Brown based on the results of similar studies with the cobalt enzyme). 

The kinetics of the binding of anions and sulphonamides to carbonic anhydrase have been measured by the stopped-flow technique. The results are consistent with a mechanism involving a pH-dependent equilibrium between two co-ordination forms of the enzyme in which anions selectively combine with the low-pH form of the enzyme whereas sulphonamides combine with the high-pH form. The effect of pH on the anion affinity correlates with the pH dependence of the spectral change associated with the cobalt(n) form of the enzyme. Further evidence on the similarity of the conformations at the active sites of the zinc and cobalt(n) forms of carbonic anhydrase has been provided by spin-labelling. A nitroxide-substituted sulphonamide was used as the spin label and its e.s.r. spectrum was found to be almost identical in the two forms of the enzyme-inhibitor complex. 

Cadmium is a toxic element (see Chapters 1,14,15) that accumulates especially in kidney and liver [4] being bound preferably to metallothionein (Chapters 6,11). On the other hand, the chemical similarity of Cd " " to Zn " is confirmed by the fact that carbonic anhydrase of marine phytoplankton contains Cd (Chapter 16), whereas the corresponding zinc enzymes are found in organisms from aU kingdoms [5] catalyzing the reversible hydration of carbon dioxide. In marine diatoms cadmium, cobalt, and zinc can functionally substitute for one another to maintain optimal growth [6]. Cadmium-carbonic anhydrase is involved in the acquisition of inorganic carbon for photosynthesis [6]. 

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