Enzyme equilibria protein structure

A relatively common feature of many problems involving molecular weight determination of biopolymers is that of association-dissociation equilibrium. Subunit structure of enzyme proteins is well recognized (1), and methods of dissociation of subunits to obtain monomer molecular weight are widely utilized (2). A previous paper described the application of an equilibrium gel partition method to the analysis of macromolecular association in a monomer-dimer case (3). The experimental parameters in a system utilizing the Sephadex series of gel filtra-... 

Stability of an enzyme is usually understood to mean temperature stability, although inhibitors, oxygen, an unsuitable pH value, or other factors such as mechanical stress or shear can decisively influence stability (Chapter 17). The thermal stability of a protein, often employed in protein biochemistry, is characterized by the melting temperature Tm, the temperature at which a protein in equilibrium between native (N) and unfolded (U) species, N U, is half unfolded (Chapter 17, Section 17.2). The melting temperature of a protein is influenced on one hand by its amino acid sequence and the number of disulfide bridges and salt pairs, and on the other hand by solvent, added salt type, and added salt concentration. Protein structural stability was found to correlate also with the Hofmeister series (Chapter 3, Section 3.4 Hofmeister, 1888 von Hippel, 1964 Kaushik, 1999) [Eq. (2.18)]. 

The mechanism can only be retained if a base very much better than water is present at the enzyme active site to deprotonate the Zn(OH2)2+ species. In fact, the structure determination reveals a histidine residue with its side chain positioned approximately halfway down the -15 A-deep cleft in the protein structure within which the Zn site is located. This arrangement could act as a proton shuttle between the Zn(OH2)2+ and external solvent water, possibly via another two water molecules also found within the cleft. As a consequence, the enhancement of ligand acidity by Zn11 is more important in the kinetic than the equilibrium sense (taken from http //www.chem.uwa.edu.aU/enrolled students/BIC sect4/sect4.2.htmll. 

Subpicosecond and picosecond motions are related to localized vibrations. According to [23], it appears that the main contribution to absorption in the spectral interval from 1 to 200 cm is caused by the hydrogen bond kinetics of the protein structural elements and of the bound water rather than by the excitation of the protein structure. Although this kind of motion primarily includes the solvent, it probably provides a viscous damping for the fast conformational fluctuations, and thus can play a certain role in the relaxation process. As noted by the authors, the most important time scales are the nanosecond and the microsecond ones. Corresponding motions determine the internal mobility in proteins, as well as in an enzyme action. Otherwise, Carreri and Gratton are sure that the motions in the millisecond-second time scale are not important for the determination of the catalytic properties of an enzyme. The authors discussed mainly the conformational fluctuations which take place near the conformationally equilibrium state of a protein globule. 

Extrusion as well as reconstitution reactions appear to be an excellent tool in the identification of the active sites in numerous iron-sulfur proteins and enzymes in which the sites cannot be directly investigated. However there are some requirements for the use of this technique, e.g. protein solubility and stability under denaturing conditions, and distinctive spectral or other characteristics permitting the differentiation of the extrusion products from the halo-protein. Structural integrity of the core during and after extrusion also appears as an important condition. Indeed, in some cases it has been observed that an equilibrium dimer-tetramer can occur. 

Note that in equilibria (2) the subscripts per and cyt are omitted where substrate S is concerned. This is obvious when the binding is measured to a solubilized transport protein, but also in the case where the enzyme is embedded in the membrane of closed vesicular structures, internal and external substrate will have equal concentrations at equilibrium (see Eig. 5). Consequently, the binding is independent of the orientation of the enzyme in the membrane. 

The family of HDAC enzymes has been named after their first substrate identified, i.e., the nuclear histone proteins. Histone proteins (H2A, H2B, H3 and H4) form an octamer complex, around which the DNA helix is wrapped in order to establish a condensed chromatin structure. The acetylation status of histones is in a dynamic equilibrium governed by histone acetyl transferases (HATs), which acetylate and HDACs which are responsible for the deacetylation of histone tails (Fig. 1). Inhibition of the HDAC enzyme promotes the acetylation of nucleosome histone tails, favoring a more transcriptionally competent chromatin structure, which in turn leads to altered expression of genes involved in cellular processes such as cell prohferation, apoptosis and differentiation. Inhibition of HDAC activity results in the activation of only a limited set of pre-programmed genes microarray experiments have shown that 2% of all genes are activated by structmally different HDAC inhibitors [1-5]. In recent years, a growing number of additional nonhistone HDAC substrates have been identified, which will be discussed in more detail below. 

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