Enzymes cobalt and

The absorption spectrum of cadmium LADH differs markedly from that of the zinc or cobalt enzyme and perhaps bears upon the nature of the metal-binding ligands (Figure 19). An intense band centered at 245 m/i with a molar absorptivity per cadmium atom of 10,200 is shown by the difference spectrum at the bottom of the figure zinc LADH is employed as the reference. Notably, the molar absorptivity of this band is nearly 14,000, close to that reported for the cadmium mercaptide chromophores of metallothionein (23). This is consistent with the hypothesis that sulfhydryl groups may serve as metal ligands in LADH. 

Azide ion, both free and bound to metal-ion complexes, also absorbs strongly in this range. A comparison between the infrared spectra of azide bound to diethylene-triamine Zn(II) and Co(II) complexes or to the cobalt-enzyme and the corresponding spectrum for the native enzyme showed that the azide ion is coordinate to the Zn(II) atom of the native enzyme. Examination of the difference spectra in the presence of both azide and CO2 showed that the bound azide sterically interferes with the binding of CO2 in the hydrophobic cavity adjacent to the Zn(ll). 

The compounds of the t/block elements show a wide range of interesting properties. Some are vital to life. Iron is an essential component of mammalian blood. Compounds of cobalt, molybdenum, and zinc are found in vitamins and essential enzymes. Other compounds simply make life more interesting and colorful. The beautiful color of cobalt blue glass, the brilliant greens and blues of kiln-baked pottery, and many pigments used by artists make use of d-block compounds. 

The introduction of redox activity through a Co11 center in place of redox-inactive Zn11 can be revealing. Carboxypeptidase B (another Zn enzyme) and its Co-substituted derivative were oxidized by the active-site-selective m-chloroperbenzoic acid.1209 In the Co-substituted oxidized (Co111) enzyme there was a decrease in both the peptidase and the esterase activities, whereas in the zinc enzyme only the peptidase activity decreased. Oxidation of the native enzyme resulted in modification of a methionine residue instead. These studies indicate that the two metal ions impose different structural and functional properties on the active site, leading to differing reactivities of specific amino acid residues. Replacement of zinc(II) in the methyltransferase enzyme MT2-A by cobalt(II) yields an enzyme with enhanced activity, where spectroscopy also indicates coordination by two thiolates and two histidines, supported by EXAFS analysis of the zinc coordination sphere.1210... 

The UV spectra of these complexes are very similar to those found in tetrahedral complexes of Co11 known to have a somewhat distorted geometry, suggesting a similar geometry in the cobalt enzyme.1383 Tetrahedral mercaptide complexes of the type [Co(SPh)4]2- were also shown to have similar absorption characteristics to those of [(LADH)Co2Co2j. This work is in complete agreement with the X-ray crystallographic studies of the native LADH, already mentioned, which shows distorted tetrahedral coordination of both the catalytic and non-catalytic zinc atoms of the enzyme. 

In order to show that the origin of this difference is not a function of the particular substrate analogue used, similar NMR relaxation studies have been performed with dimethyl sulfoxide (DMSO)1401 since the crystal structure of the enzyme-NADH-DMSO ternary complex is well resolved.1366 From the relaxation data, the distance between the methyl protons of DMSO and Co11 was calculated to be 8.9 0.9 A, again too great for direct coordination of the sulfoxide group to the metal ion. Since the cobalt enzyme appears to be functionally similar to the native enzyme, the difference is unlikely to be a direct result of substitution. One possibility is that there may actually be a difference between the solution and crystalline structure of the enzyme ternary complex, particularly since it is well established that the crystalline enzyme is 1000 times less active than in solution.1402... 

There has been some uncertainty concerning the metal content of alkaline phosphatase and the role of zinc in the catalytic process. Early measurements by Plocke et al. (36, 50) showed that there were 2 g-atoms per dimer. The zinc requirement for enzymic activity was demonstrated by the inhibition of the enzyme with metal binding agents in accord with the order of the stability constants of their zinc complexes. It appears that in some cases (EDTA) zinc is removed from the enzyme and in other cases (CN) the ligand adds to the metalloprotein. A zinc-free inactive apoenzyme was formed by dialysis against 1,10-phenanthro-line. Complete activity was restored by zinc only zinc, cobalt, and possibly mercury produce active enzyme. 

The origin of these pKa values has been explored by H NMR studies on cobalt LADH,546 which suggest that the pKa value of 9.0 in Co(c)2Zn(n)2-LADH is due to the S-NH group of His-67, one of the ligands. In the complex with NAD+ the same group shows a pH-dependent shift without deprotonation, with a p a of 8.3 0.2, which is suggested to correspond to the pKa of 7.6 obtained from kinetic data on the native enzyme, and to be due to coordinated water. The suggestion that His-67 is involved in the acid-base equilibria is novel and requires substantiation. 

An enzyme cofactor can be either an inorganic ion (usually a metal cation) or a small organic molecule called a coenzyme. In fact, the requirement of many enzymes for metal-ion cofactors is the main reason behind our dietary need for trace minerals. Iron, zinc, copper, manganese, molybdenum, cobalt, nickel, and selenium are all essential trace elements that function as enzyme cofactors. A large number of different organic molecules also serve as coenzymes. Often, although not always, the coenzyme is a vitamin. Thiamine (vitamin Bj), for example, is a coenzyme required in the metabolism of carbohydrates. 

If our postulates are correct the most interesting feature of P-450 is the manner in which the protein has adjusted the coordination geometry of the iron and then provided near-neighbour reactive groups to take advantage of the activation generated by the curious coordination. Vallee and Williams (68) have observed this situation in zinc, copper and iron enzymes and referred to it as an entatic state of the protein. It is also apparent that some such adjustment of the coordination of cobalt occurs in the vitamin B12 dependent enzymes. As a final example we have looked at the absorption spectra of chlorophyll for its spectrum is in many respects very like that of a metal-porphyrin. This last note is intended to stress the features of chlorophyll chemistry which parallel those of P-450. 

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). 

Some efforts have been made to interpret the spectroscopic and magnetic properties of cobalt enzymes in terms of coordination geometry and chemical identity of ligands. The basis of these attempts is a comparison with the corresponding properties of low-molecular weight complexes of known structure. A brief summary of relevant data on some models is given in the following section. 

Measurements of the magnetic susceptibility (58) of the cobalt enzyme (Table 5) show that the metal ion is bound as high-spin Co (II). The intensity of the visible absorption makes an octahedral coordination, as well as tetragonal distortions thereof, very unlikely. In the combination with CN, the Co(II) enzyme exhibits the spectral features of tetrahedral model complexes with regard to intensity as well as structure both in the visible and the near-infrared wavelength regions (Fig. 8). The width of the near-infrared band (cf. 20) indicates that the deviation... 

Zinc(II) and Co(II) are the only cations found to reactivate apophos-phatase to any appreciable extent (120). The Co(II) enzyme follows the same formal mechanism as the native enzyme, but has a lower specific activity (113, 121). It lacks the phosphotransferase activity (113, 119, 121) observed for the native enzyme, for example in Tris buffers. This was taken to imply that the lower activity of the cobalt enzyme is due to a lower rate of phosphorylation, so that this step becomes rate-limiting also below f>H 7 (113). Stopped-flow experiments by Gottesman etal. (121) show, however, that a very fast burst of -nitrophenol occurs in the cobalt alkaline phosphatase-catalyzed hydrolysis of -nitrophenyl phosphate over a wide pH region. These results strongly suggest that a step subsequent to the phosphorylation is rate-limiting in this metal derivative. 

On the other hand, alkaline phosphatase may have two equivalent active sites which are coupled so that, normally, only one can operate at a time. This seems an attractive alternative for an enzyme consisting of two identical subunits. In a preliminary paper, Lazdunski et al. (125) report the covalent incorporation of two phosphates into the zinc enzyme as well as the cobalt enzyme, at >H<4. At these low pH values, the free enzyme generally loses its metal ions and dissociates into monomeres (109). However, if these results are corroborated after the performance of proper controls, and if both phosphates are linked to specific amino acid residues in the enzyme, conditions may have been found for the uncoupling of active sites in alkaline phosphatase. 

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