Specificity of the Substrate

In the preceding chapters — with the exception of the discussion of different flavoproteins — no such discussion was started regarding the specificity of the superoxide anion as the sole substrate for erythrocuprein. In this context let us recall some basic principles of the chemical and physical properties of oxygen (for extensive reviews see Refs. (172— 178 a)). 

The O2 molecule can be considered as a stable biradical. In contrast to the N 2 molecule, two rr -antibonding molecular orbitals are occupied according to Hund s rule, each by one electron. Both electrons have the same spin direction. This binding situation represents the ground state of the electronic triplet expressed as  

Upon excitation of this triplet ground state, two metastable singlet states 

Thanks to its electron affinity, oxygen is able to accept up to four electrons (Fig. 29). 

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The catalysis takes place in a specific region of the enzyme named the active site or catalytic cavity. This active site involves those amino acid residues (i.e., side chains) directly implicated in the mode of binding and the specificity of the substrate, as well as in the catalytic process itself. 

The effect of donor ligands, usually expected to enhance the reactivity of organolithi-ums, was studied in the case of the addition of PhLi to ( )-cinnamaldehyde in THF, under conditions that lead to 1,3-diphenylpropanone. TMEDA and HMPT were found to decelerate the reaction and with an [HMPA] [PhLi] ratio >4, the reaction became almost completely inhibited. These results show the complexity of solvation effects and the specificity of the substrate-reagent-ligand-solvent interactions.222... 

The source of the peptidase will depend on the aim of the assay i.e., one may wish to measure catalytic activity in tissue or plasma samples, in which case specificity of the substrate is of prime importance. Conversely, if the assay is to be used in the development of peptidase inhibitors, the enzyme should be present in as pure a form as possible. We routinely express a recombinant form of ECE-1 in Chinese hamster ovary cells, and generally use the wild-type, membrane-bound form of the enzyme in crude membrane preparations. However, we have also designed a soluble, secreted form of ECE-1 containing a hexahistidine tag this allows purification on a nickel affinity resin and eliminates potential problems involved with the use of crude, particulate membranes. 

The use of tissue slices for experiments on histidine decarboxylation introduces the additional problem of the access of substrate, co-enzyme and inhibitors into the cells. In this connection, it should be noted that in practice the specificity of an enzyme within a cell may be increased by the specificity of the substrate-transporting system. Similar considerations apply to the in vivo inhibition of histidine decarboxylases there is, however, the additional possibility of modifying production of the apo-enzyme either by restricting the supply of amino acids or by altering the hormonal state of the animal. 

The classical separation of specificity and catalysis may not be valid. Often the specificity of the substrate binding process, which may be described by some sort of lock and key model is treated as a separate process from the catalytic rate acceleration which is often erroneously explained in solely chemical terms. In fact, many enzymes apparently exhibit their specificity in their maximal rates of catalysis rather than in substrate binding. 

P-Galactosidase. /8-galactosidase itself has been made in vitro and subjected to sedimentation analysis. Depending on how much enzyme was synthesized in vitro, the activity sedimented as a monomer or dimer (7.4 S or 10.2 S respectively). Upon further concentration, the in vitro product formed tetramers (16.6 S) similar to the native enzyme (Zubay and Chambers, I969). The nature of the enzymatic activity of the in vitro enzyme is unequivocally defined by the specificity of the substrate. Further purification of /ff-galactosidase synthesized in vitro was achieved and a structural and kinetic analysis of both the in vitro enzyme and the natural enzyme showed identity (Chambers and Manley, 1973). 

The nature of the real surface depends on its formation, handling, and storage history. In order to have reproducible film properties, the substrate surface must be reproducible. This reproducibility is attained by careful specification of the substrate material, incoming inspection procedures, surface preparation, and appropriate handling and storage of the material. 

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