Metalloenzyme specific types

Superoxide dismutases (SODs) are a family of cytosolic metalloenzymes that specifically remove (reviewed by Omar etal., 1992). SOD distribution within the body is ubiquitous, being found in erythrocytes as well as most organs and cell types. Three distinct mammalian SOD forms exist CuZnSOD, MnSOD and extracellular SOD (EC-SOD). Their amino-acid sequences differ as well as the transition metals at their active sites. Rheumatoid synovial fluid contains low levels of SOD activity and hence little protection from ROM generated by infiltrating PMNs (Blake etcU., 1981). Furthermore, leucocytes from patients with RA are deficient in MnSOD, which might promote the extracellular leakage of O2 (Pasquier et al., 1984). 

To establish that a-D-mannosidase is a metalloenzyme, the proportion of bound zinc in the active molecule must first be determined. This experiment has been performed for the enzyme from jack-bean meal.27 Two types of preparation were employed. The first was purified, throughout, at pH 8 without any addition of Zn2+ (see Section 11,4 p. 409). The second enzyme preparation was purified in the presence of added Zn2+ at pH 5 up to stage 5 of the original procedure (see Table V p. 410). It was then freed from unbound Zn2+ by dialysis against glycine buffer of pH 8, and passed through a column of Sephadex G-100 in the same buffer. As may be seen from Table IX, the final specific activity was, in each instance, slightly less than that shown in Table V. 

We have chosen to discuss enzyme modification of proteins in terms of changes in various functional properties. Another approach might have been to consider specific substrates for protease action such as meat and milk, legumes and cereals, and the novel sources of food protein such as leaves and microorganisms ( ). Alternatively, the proteases themselves provide categories for discussion, among which are their source (animals, plants, microorganisms), their type (serine-, sulfhydryl-, and metalloenzymes), and their specificity (endo- and exopeptidases, aromatic, aliphatic, or basic residue bond specificity). See Yamamoto (2) for a review of proteolytic enzymes important to functionality. 

It is possible in many metalloenzymes to substitute the native metal ion with another first transition ion either by removing the original ion with chelating agents or by exchange dialysis. In addition to certain interesting physical properties that can be studied by this method, an opportunity is provided to discover what kind of specificity for catalysis resides in the electronic properties of the metal ion. Studies of this type have been conducted with several metalloenzymes. 

The specificities of the various digestive exo- and endopep-tidases suggest that they act synergistically to fulfill a major nutritional function. The concerted action of trypsin, chy-motrypsin, pepsin, and carboxypeptidases A and B facilitate and ensure formation of essential amino acids. The chemical characteristics and metalloenzyme nature of two bovine exopeptidases, lens aminopeptidase and pancreatic carboxy-peptidase A, indicate similarities in their mechanisms of action. However, the aminopeptidase exhibits an unusual type of metal ion activation not observed unth carboxy-peptidase. Chemical and physicochemical studies reveal that the latter enzyme has different structural conformations in its crystal and solution states. Moreover, various kinetic data indicate that its mode of action toward ester substrates differs from that toward peptide substrates. The active site metal atom of carboxypeptidase figures prominently in these differences. 

It is notable that reaction does not occur between free maleate ion and free 1,2-ethanediamine, although it does occur (but only very slowly) with maleate diester and 1,2-ethanediamine. Acceleration of the reaction of the maleate resulting from coordination to a metal ion is significant, and lies in the range 106-1010. Accelerations of this size are common for reactions of coordinated nucleophiles. The observation of stereospecificity and large accelerations suggests that these types of reactions may be relevant to the modes of action of certain metalloenzymes, where reactions are very rapid, specific and stereoselective. Such reactions are met in Chapter 8. 

An exhaustive review of all of the types of reactions that are catalyzed by metal-requiring enzymes and the specific functions of these metals, as currently understood, is beyond the scope of this chapter. To complicate this general area of investigation, even within a single group of enzymes, the metal ions may play different roles in the catalytic processes for reaction-related enzymes. Not all enzymes of a specific class necessarily require a cation for activity. In some cases, the roles of the cation may be substituted by specific amino acid residues in the protein. A classic example of such a case is the muscle and yeast fmctose-bisphosphate aldolases. The muscle enzyme catalyzes the aldol condensation using a Schiff base intermediate to activate the substrate, whereas the yeast enzyme is a Zn +-metalloenzyme (1). The cation appears to serve as the electrophile in the activation of the substrate for the same reaction. 

In concluding this chapter, it should be noted that we have presented various models of enzyme mechanisms in which metal ions participate. We have seen that reactions catalyzed by metalloenzymes or enzymes activated by metal ions span a remarkably broad spectrum of reaction types. Of course many facets such as the remarkable speed and specificity of enzyme catalysis have not received complete explanation on the basis of model system chemistry. However, the missing link may be found at the point where biological molecules deviate from the model systems. Here, perhaps the most interesting chemical features will be found (258,259). 

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