Enzymes, quaternary structure

Understanding the action mechanism of flavoenzymes heavily relies on the combination of different chemical tools and techniques. First, it is of utmost importance to have a pure and stable (recombinant) protein. Size exclusion chromatography will provide information about the enzyme quaternary structure, and mass spectrometry can establish posttranslational modifications. For a detailed insight into the protein structure, well-diffracting crystals are needed to determine the X-ray structure. 

The basic kinetic properties of this allosteric enzyme are clearly explained by combining Monod s theory and these structural results. The tetrameric enzyme exists in equilibrium between a catalytically active R state and an inactive T state. There is a difference in the tertiary structure of the subunits in these two states, which is closely linked to a difference in the quaternary structure of the molecule. The substrate F6P binds preferentially to the R state, thereby shifting the equilibrium to that state. Since the mechanism is concerted, binding of one F6P to the first subunit provides an additional three subunits in the R state, hence the cooperativity of F6P binding and catalysis. ATP binds to both states, so there is no shift in the equilibrium and hence there is no cooperativity of ATP binding. The inhibitor PEP preferentially binds to the effector binding site of molecules in the T state and as a result the equilibrium is shifted to the inactive state. By contrast the activator ADP preferentially binds to the effector site of molecules in the R state and as a result shifts the equilibrium to the R state with its four available, catalytically competent, active sites per molecule. 

Type II copper enzymes generally have more positive reduction potentials, weaker electronic absorption signals, and larger EPR hyperfine coupling constants. They adopt trigonal, square-planar, five-coordinate, or tetragonally distorted octahedral geometries. Usually, type II copper enzymes are involved in catalytic oxidations of substrate molecules and may be found in combination with both Type I and Type III copper centers. Laccase and ascorbate oxidase are typical examples. Information on these enzymes is found in Tables 5.1, 5.2, and 5.3. Superoxide dismutase, discussed in more detail below, contains a lone Type II copper center in each of two subunits of its quaternary structure. 

Table 5.2 contains data about selected copper enzymes from the references noted. It should be understood that enzymes from different sources—that is, azurin from Alcaligenes denitrificans versus Pseudomonas aeruginosa, fungal versus tree laccase, or arthropodan versus molluscan hemocyanin—will differ from each other to various degrees. Azurins have similar tertiary structures—in contrast to arthropodan and molluscan hemocyanins, whose tertiary and quaternary structures show large deviations. Most copper enzymes contain one type of copper center, but laccase, ascorbate oxidase, and ceruloplasmin contain Type I, Type II, and Type III centers. For a more complete and specific listing of copper enzyme properties, see, for instance, the review article by Solomon et al.4... 

Figure 8.10 The quaternary structure of proteins. The enzyme lactate dehydrogenase (EC 1.1.1.27) has a relative molecular mass of approximately 140 000 and occurs as a tetramer produced by the association of two different globular proteins (A and B), a characteristic that results in five different hybrid forms of the active enzyme. The A and B peptides are enzymically inactive and are often indicated by M (muscle) and H (heart). The A4 tetramer predominates in skeletal muscle while the B4 form predominates in heart muscle but all tissues show most types in varying amounts.

In addition to the binding of substrate (or in some cases co-substrates) at the active site, many enzymes have the capacity to bind regulatory molecules at sites which are usually spatially far removed from the catalytic site. In fact, allosteric enzymes are invariably multimeric (i.e. have a quaternary structure) and the allosteric (regulatory) sites are on different subunits of the protein to the active site. In all cases, the binding of the regulatory molecules is non covalent and is described in kinetic terms as noncompetitive inhibition. 

The enzyme responsible for this topping-up ATP in active muscle is CK. CK is found in high concentration in muscle cells, both free within the sarcoplasm and also associated with membranes of mitochondria and the sarcoplasmic reticulum. Structurally, creatine kinase is a dimeric enzyme of B and/or M subunits, each of about 40 kDa. Three quaternary structure isoenzyme forms arise CK-MM, CK-BB and CK-MB. The predominant form in all muscles is CK-MM, but cardiac muscle also contains a significant amount of CK-MB and this isoenzyme can be used as a specific marker of myocardial damage (see Case Notes at the end of this chapter). 

Pressure may cause several changes in enzymes, as well as some changes which are not directly associated with the catalytic process. These changes may include conformational changes and subunit dissociation-association processes. Pressures above 4000 bar may induce conformational changes to such an extent that the enzyme in effect becomes irreversibly denatured. These are dealt with in the next section. In this section we will deal with lower pressures and reversible processes, namely, interactions between subunits in quaternary structures. For most multimeric enzymes, the maintenance of... 

Recently it has been shown that when a protein molecule contains two or more sub-units, which may or may not be identical, the quaternary structure or relative positions of the sub-units is important for function. Many regulatory enzymes are examples of this type of protein 26, 27). 

The fact that ATP and CTP bind to the same site follows from the observation that adding ATP to the inhibited enzyme by CTP reduces or reverses the inhibition, presumably because ATP competes with CTP for the same site. The fact that CTP binds to an allosteric site (i.e., it is not a competitive inhibitor) follows from the so-called desensitization effect. Addition of mercurials [e.g., p-mercuribenzoate (PMB)] reduces or eliminates the inhibition by CTP. However, it has no effect on the enzymatic activity of ATCase, presumably because the mercurials affect the regulatory subunits but not the catalytic site. As for the mechanism of cooperativity (both positive and negative), it is known that CTP does induce changes in the quaternary structure of the enzyme. 

The diversity among catalases, evident in the variety of subunit sizes, the number of quaternary structures, the different heme prosthetic groups, and the variety of sequence groups, enables them to be organized in four main groups the classic monofunctional enzymes (type A), the catalase-peroxidases (type B), the nonheme catalases (type C), and miscellaneous proteins with minor catalatic activities (type D). 

The third type of inhibition is called allosteric inhibition, and is particularly important in the control of intermediary metabolism This refers to the ability of enzymes to change their shape (tertiary and quaternary structure, see Section 13.3) when exposed to certain molecules. This sometimes leads to inhibition, whereas in other cases it may actually activate the enzyme. The process allows subtle control of enzyme activity according to an organism s demands. Further consideration of this complex phenomenon is outside our immediate needs. 

Huebner, G. Koenig, S. Koch, M.H.J. Hengstenberg, W. Influence of Phosphoenolpyruvate and Magnesium Ions on the Quaternary Structure of Enzyme I of the Phosphotransferase System from Gram-Positive Bacteria. Biochemistry, 34, 15700-15703 (1995)... 

With the exception of a small group of catalytic RNA molecules (Chapter 26), all enzymes are proteins. Their catalytic activity depends on the integrity of their native protein conformation. If an enzyme is denatured or dissociated into its subunits, catalytic activity is usually lost. If an enzyme is broken down into its component amino acids, its catalytic activity is always destroyed. Thus the primary, secondary, tertiary, and quaternary structures of protein enzymes are essential to their catalytic activity. 

Allosteric enzymes are generally larger and more complex than nonallosteric enzymes. Most have two or more subunits. Aspartate transcarbamoylase, which catalyzes an early reaction in the biosynthesis of pyrimidine nucleotides (see Fig. 22-36), has 12 polypeptide chains organized into catalytic and regulatory subunits. Figure 6-27 shows the quaternary structure of this enzyme, deduced from x-ray analysis. 

Most known thiamin diphosphate-dependent reactions (Table 14-2) can be derived from the five halfreactions, a through e, shown in Fig. 14-3. Each halfreaction is an a cleavage which leads to a thiamin- bound enamine (center, Fig. 14-3) The decarboxylation of an a-oxo acid to an aldehyde is represented by step b followed by a in reverse. The most studied enzyme catalyzing a reaction of this type is yeast pyruvate decarboxylase, an enzyme essential to alcoholic fermentation (Fig. 10-3). There are two 250-kDa isoenzyme forms, one an a4 tetramer and one with an ( P)2 quaternary structure. The isolation of ohydroxyethylthiamin diphosphate from reaction mixtures of this enzyme with pyruvate52 provided important verification of the mechanisms of Eqs. 14-14,14-15. Other decarboxylases produce aldehydes in specialized metabolic pathways indolepyruvate decarboxylase126 in the biosynthesis of the plant hormone indoIe-3-acetate and ben-zoylformate decarboxylase in the mandelate pathway of bacterial metabolism (Chapter 25).1243/127... 

The structure of proteins, as with the structure of carbohydrates, has various levels—primary, secondary, tertiary and quaternary structure. The tertiary and quaternary structures and their subtleties are most important in the biological function of the molecule. Consider an enzyme (a protein-based catalyst)—its structure allows the binding of specific molecules which then react catalytically to give products. Conversely, enzymes are very susceptible to environmental conditions which alter their tertiary structure. 

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