Chemistry and Biology of Proteins - F. Haurowitz 1953

Proteins with Enzymatic Properties
Physicochemical Properties of Enzymes

Certain Enzymes dissolved in gastric and intestinal juices, Blood serum, and other Body Fluids are typical globulins soluble in dilute neutral salt solutions. Similar enzymes present in Cells can be extracted using the same Methods applied to isolate other soluble cellular Proteins. Brief high-speed centrifugation removes Cell/35.html">Mitochondria and other cell granules containing specific enzymes from these turbid extracts. Other Enzymes are localized in microsomes, which sediment from the extracts only after prolonged high-speed centrifugation. Finally, a fraction of the enzymes is associated with insoluble structural cellular proteins. While most hydrolytic enzymes, such as Pepsin, Trypsin, or urease, are soluble Globular proteins, oxidative enzymes like succinoxidase and cytochrome oxidase are located primarily in cellular particulates [12]. The Cleavage of ATP by Myosin indicates that Fibrillar Proteins can also exhibit enzymatic activity [13].

The colloidal (insoluble) state of enzymes within cells appears to be essential for their biological function. If catalytically active low-molecular-weight prosthetic groups existed in a free state within The Cell, they could easily diffuse across cell membranes and thus escape from the cells. This loss is prevented by the binding of small molecules, such as heme or riboflavin, to protein molecules.

Another consequence of the colloidal state of enzymes is The formation of phase boundaries between the aqueous phase and the insoluble (colloidal) protein phase. The heterogeneous Nature of the medium housing the enzymes frequently facilitates their catalytic action. For instance, hemin, which exhibits pronounced lipoxidase activity in a heterogeneous medium (Water-in-oil emulsion), completely loses this activity when the emulsion is converted into a homogeneous solution by adding ethyl alcohol or Bile salts [14].

All Enzymes can be divided into two categories: those lacking prosthetic groups and those containing catalytically active non-protein groups (e.g., heme, riboflavin, copper, or thiamine pyrophosphate). As a rule, enzymes participating in oxidation-reduction reactions belong to the second category, whereas hydrolytic enzymes apparently contain no prosthetic groups. However, this latter Conclusion must be approached with caution. Although amino acid analyses of these enzymes have shown that they consist of at least 99% Amino Acids, this does not yet provide definitive proof that these enzymes completely lack any kind of prosthetic group. If an enzyme molecule has a Molecular Weight of 50,000, 1% amounts to 500, meaning that the enzyme molecule could still contain a hitherto unknown prosthetic group. Studies on peptidases appear to support the view that even highly purified preparations of single-component enzymes contain a prosthetic group. These investigations have demonstrated that The activity of certain peptidases depends on the presence of Metal Ions [15] such as cobalt, zinc, manganese, magnesium, or calcium in their composition. For instance, it has been established that carboxypeptidase [16] and alkaline phosphatase from Kidney tissue [17] are complexes of protein and magnesium.

Following the suggestion of several researchers, the protein moiety of an enzyme has been designated as the apoenzyme, while the prosthetic group has been termed the coenzyme. In most cases, the coenzyme can be separated from the apoenzyme, for example, by dialysis against an acid. Like other proteins, apoenzymes are denatured by heat. It is now considered established that apoenzymes, once separated from their Coenzymes, are less resistant to Denaturing Agents than the intact enzyme molecule. On this basis, one can conclude that the binding of the coenzyme stabilizes the apoenzyme. In their free state, coenzymes are thermostable substances that do not lose their properties upon boiling in solution; consequently, recombining a coenzyme with its apoenzyme restores the full activity characteristic of the native enzyme molecule.

Although in complex enzymes the functional group is undoubtedly the coenzyme, the presence of the apoenzyme is absolutely essential for full enzymatic activity. An isolated prosthetic group generally exhibits only very weak catalytic activity. Upon binding with the apoenzyme, catalytic activity increases, sometimes more than a thousandfold compared to that of the free prosthetic group. This is partly due to the high molecular weight of the resulting complex and partly to the specific action of the apoenzyme, which cannot be replaced by any other protein. All apoenzymes exhibit Specificity, manifested in their ability to combine exclusively with specific coenzymes, whereupon the bound coenzyme catalyzes only specific, designated reactions.

¹ In a paper published in 1951 (Biokhimiya, 16, 81, 1951), S. M. Bresler and N. A. Rosentsveig concluded that trypsin and Chymotrypsin contain metal ions in their active complexes: magnesium in chymotrypsin, and chromium in trypsin. According to these authors, the magnesium in chymotrypsin is tightly bound and is not removed by dialysis, whereas the chromium in trypsin is loosely bound and can be removed by dialysis as well as replaced by magnesium, while the catalytic activity of trypsin is preserved. — Ed. note.

While apoenzymes can combine with only one specific coenzyme, a single coenzyme has The ability to bind with various apoenzymes. Thus, for instance, substances as markedly different in their catalytic properties as Hemoglobin, catalase, peroxidase, and cytochrome c share the same protoheme as their prosthetic group, differing from one another solely in their protein components. Similarly, certain dehydrogenases, such as lactic dehydrogenase and phosphoglyceraldehyde dehydrogenase, share the same prosthetic group—a pyridine nucleotide—yet possess different apoenzymes [18].

The Nature of the bond between the coenzyme and the apoenzyme can vary. In some enzymes, the coenzyme is linked to the apoenzyme via covalent bonds, while in others it is bound by electrovalent linkages or Structure/103.html">Van der Waals forces. We cannot yet satisfactorily explain what determines the nature of these bonds or how the apoenzyme confers specificity upon the action of the prosthetic group.