Quaternary structures dissociation

Positive modulation (protection) by substrate has been explained in terms of the stiffness of the enzyme structure that it promotes (Villaume et al. 1990). Negative modulation (destabilization) has also been reported for substrate (Illanes et al. 1998a) and other catalytic modulators (Alvaro et al. 1991). This behavior can be explained in terms of the promotion of quaternary structure dissociation (Misset 1993) and alteration of the oxidation stage in the active site of the enzyme (Bourdillon et al. 1985). 

Associations of two or more protein molecules lead to the so-called quaternary structures. At infinite dilution and with suitable solvents, quaternary structures dissociate into subunits. The thermodynamic stability of quaternary structures is often so high that no noticeable dissociation... 

The forces that stabilize quaternary structure have been evaluated for a few proteins. Typical dissociation constants for simple two-subunit associations... 

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... 

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. 

The enzyme contains six Zn2+ per molecule, two per R subunit. The zinc is not required for catalytic activity, but is essential for the maintenance of the quaternary structure. The structure has been determined to a resolution of 2.8 A in the presence and absence of CTP.528 The zinc-binding site is located in the C-terminal region of the R chains, and involves four cysteinyl residues, with tetrahedral geometry. The zinc domain represents the major site of interaction between the R and C chains, explaining the importance of zinc for the association of the subunits and the dissociative effect of mercurial reagents. When E. coli is grown in a zinc-deficient medium, some 70% of the enzyme is found to be dissociated into subunits.529... 

The kinetics of binding of N02 to the four subunit methemoprotein, methemo-globin (metHb) were investigated. The rate constants for fast and slow reactions were comparable with literature values.325 The thermally derived activation parameters indicated that the reactions all proceed by a dissociative mechanism. It was predicted that hydrostatic pressure could affect the compressibility of the four subunits and quaternary structure of metHb, and therefore volume parameters of reliable value for the reactions of metMb would not be obtained. 

The CD spectra in the far-UV region revealed that the pro-tyrosinase and acid-activated tyrosinase had similar secondary structures (Figure 40). On the other hands, the CD signal of acid-activated tyrosinase in the range of 280-290 nm, indicating that the tertiary and/or quaternary structure of the protyrosinase was changed by acid-treatment. On the basis of these results, we deduce that the intersubunit polar interaction is disrupted at pH 3.0, and that the tetrameric protyrosinase dissociates to dimmers. 

Honda and Tokushige reinvestigated the effect of temperature and monovalent cations on the quaternary structure of tryptophanase using the HPLC gel-filtration analysis.28 In contrast to the above-mentioned data, they found that in the absence of K+ or NH4+ ions the tetrameric holoenzyme undergoes dissociation into dimers and inactivation at 5°C. Their results also indicate that formation of the active holoenzyme from the apoenzyme and PLP proceeds in two steps the inactive tetrameric species are first formed then converted to the active species only the second step requires the presence of K+ or NH4+ ions (Fig. 9.3)... 

Proteins that possess a quaternary structure are composed of several separate polypeptide chains held together by noncovalent interactions. When such proteins are examined under dissociating conditions (e.g., 8 M urea to weaken hydrogen bonds and hydrophobic interaction, 1 m/lf mercaptoethanol to disrupt disulfide bonds), the molecular weight of the component polypeptide chains can be determined. By comparison with the native molecular weight, it is often possible to determine how many polypeptide chains are involved in the native structure. 

The detergent SDS causes the dissociation of quaternary structures and allows the determination of molecular weight of the component subunits. The data suggest that the enzyme comprises four identical subunits of Mr = 40,000, yielding a tetramer of Mr = 160,000. 

The quaternary structure of a protein can be largely determined by careful molecular weight determination of the native protein, followed by dissociation of the protein into its constituent polypeptide chains with denaturing agents. If nonidentical subunits are found, they must be separated from each other. Then the molecular weight of the isolated subunits must be determined, usually under dena-... 

MALDI has also been successfully used to study a number of intact non-covalent complexes such as protein quaternary structures [132-135]. However, MALDI has not yet contributed widely to the study of these complexes because it requires the crystallization of the sample with the matrix. Furthermore, the energy deposited to desorb the ions is not clearly known. In consequence, MALDI generally induces dissociation of the non-covalent interactions and leads to the formation of non-specific aggregates. Nevertheless, weak non-covalent interactions can survive during the MALDI process to allow the direct detection of intact complexes if specific methods, which have been developed to preserve these interactions, are followed [136],... 

The major soy proteins, the 7S and the 11S globulins are characterized by complex quaternary structures easily undergoing association-dissociation reactions. Salt has a unique stabilizing effect on the quaternary structure of both the 7S and the 11S globulins, which influences most physical properties of soy proteins. 

The mass of a protein can be directly determined by sedimentation equilibrium, in which a sample is centrifuged at relatively low speed so that sedimentation is counterbalanced by diffusion. The sedimentation-equilibrium technique for determining mass is very accurate and can be applied under nondenaturing conditions in which the native quaternary structure of multimeric proteins is preserved. In contrast, SDS-polyacrylamide gel electrophoresis (Section 4.1.4) provides an estimate of the mass of dissociated polypeptide chains under denaturing conditions. Note that, if we know the mass of the dissociated components of a multimeric protein as determined by SDS-polyacrylamide analysis and the mass of the intact multimeric protein as determined by sedimentation equilibrium analysis, we can determine how many copies of each polypeptide chain is present in the multimeric protein. 

Figure 6. Proposed quaternary structures of rhSCF dimer and disulfide-linked dimer. A, SDS-dissociable rhSCF dimer with topology similar to M-CSF. B, disulfide-linked dimer having all disulfides at the dimer interface. C, disulfide-linked dimer containing A and D helices swapped between subunits (distinguished as shaded and unshaded helices). The four helical structure (A-D helices) was derived from that proposed by Bazan (27).

Quaternary structure may influence the activity of enzymes. Some enzymes are active only in their quaternary state and become inactive wTien split into smaller units. Other enzymes are inactive in the quaternary state and are activated only when they are dissociated to form monomeric state. 

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