Jacqueline A. Reynolds

Binding of Organic Ions by Proteins

Publisher Summary nThis chapter describes the binding of organic ions by proteins. Although proteins differ with respect to whether they show measurable tendencies to bind small anions, all proteins that have been examined bind large organic anions (the longer-chain fatty acids, ionic detergents, and such aromatic compounds as dyes) to some extent. In general, within a homologous ligand series, the binding affinity increases with the size of the ion. Under some conditions, the binding of these large ions results in precipitation of the complex formed. However, binding and precipitation do not go together in any simple way. The binding of anions by those proteins that have been investigated in detail cannot be expressed as the result of simple multiple equilibria that involve sets of identical binding groups, modified only by the electrostatic effects resulting from changes in charge as the protein interacts with different numbers of ions. These difficulties may be partly because of the fact that detailed ion-binding measurements on soluble proteins have been made on a very small number of proteins.

FORMATION AND LOCALIZATION OF THE ALKALINE PHOSPHATASE OF ESCHERICHIA COLI

The alkaline phosphatase of Escherichia coli is a nonspecific phosphomonoesterase that enables the bacterial cell to utilize organic phosphates when these are the only source of phosphate present in the medium. The enzymatic properties, the structure, and the genetics of this protein have been studied in several laboratories. A partial summary of the results of these investigations follows: 1. The enzyme is a stable, globular, zinc metalloprotein (molecular weight = 86,000) composed of two identical subunits.14 2. The protein is localized exterior to the cell cytoplasm in the periplasmic ~pace.~*6 3. The structural gene is a single cistron; mutations in this gene can lead to inactive proteins (CRM) that cross-react with antienzyme and/or antisubunit antibodies. 1,8 4. In exponentially growing cultures of E. coli K12, formation of alkaline phosphatase is repressed and enzyme appears only when the growth medium becomes depleted of inorganic pho~phate .~ Mutations in any one of three distinct gene loci lead to cells (constitutives) that form enzyme even when high levels of inorganic phosphate are present in the growth medium.IO 5 . The enzyme can act as a phosphotransferase, and a phosphorylated enzyme has been is0lated.~~J3 6. Enzymatic activity varies with zinc content (3 to 4 Z n + + per dimer have been measured for the pure enzyrneI4). Removal of zinc abolishes enzymatic activity. Only one catalytically active site per dimer can be measured by equilibrium dialysis against inorganic phosphate, a competitive inhibitor.14 We have been studying the relation of structure to function of the E. coli alkaline phosphatase with particular interest in describing the conformational changes that occur in the protein when the unfolded polypeptide chains are assembled into an ordered, compact, enzymatically active dimer. One aim of this study has been to determine how this enzyme becomes localized to the exterior of the cell cytoplasm. In this paper, we review the results of several experimental approaches that have been carried out to investigate the formation and localization of alkaline phosphatase.

Metal-Ion Binding

Publisher Summary nThis chapter describes metal–ion binding. Two general classes of proteins are considered for describing metal–ion binding. These are (1) systems in which the metal ion occupies a small number of very high energy sites and is essential for the biological function of the macromolecule (e.g., alkaline phosphatase, carboxypeptidase) and (2) systems in which metal binds reversibly to specific amino acid residues in the polypeptide chain but is not required for biological activity and indeed may even impair protein function or disrupt protein structure. Metal ions, like protons, share electron pairs from the donor atoms of a ligand molecule and, thus, form partially covalent bonds with characteristic heats of formation. This type of binding is distinguished from binding to proteins of neutral molecules or large organic ions such as detergents, where the large binding forces are primarily entropie in origin. All metal ions have sets of characteristic coordination numbers that represent the number of hybrid bonds available for ligands. The binding of metal ions to proteins can be measured by equilibrium dialysis.

Hydrogen-Ion Equilibria

Publisher Summary nThis chapter discusses hydrogen–ion Equilibria. It discusses the ambiguities and uncertainties that sometimes occur, instead the model systems in terms of which hydrogen–ion equilibria of proteins. While all such groups would be expected to be accessible to solvent and ionize with normal pKs in linear unstructured macromolecules, these groups do not always titrate normally in proteins. An important object of studies of hydrogen–ion equilibria of biological macromolecules is to determine as to which of the prototropic residues are normal in that they have pKs similar to model compounds and are in some manner abnormal because of either steric inaccessibility or specific interactions with other charged groups in the macromolecule. The isoionic point is the pH of a protein solution that contains only protein and ions arising from them dissociation of the solvent. A solution of protein in deionized water may be made isoionic by passage through an ion-exchange resin or exhaustive dialysis against deionized water. The resulting solution may be very close to isoelectric if the true isoelectric point lies between pH 4 and pH 10 and if the protein combines only with hydrogen and hydroxyl ions.

Thermodynamics and Model Systems

Publisher Summary nThis chapter presents the mathematical description of complex formation, the thermodynamic analysis of binding data, and various theoretical models that can be used to describe the phenomena of small molecule–macromolecule interactions. The experimental quantities of interest in the study of multiple equilibria are the molal binding ratio, defined as the average number of moles of ligand bound per mole of protein, and the ligand concentration in equilibrium with the ligand complex. These two experimentally determined quantities permit one to calculate the association constants of ligand for the site or sites on the protein as well as the free energy of the complex formation. When binding is measured as a function of temperature, other thermodynamic quantities such as enthalpy and entropy of binding can also be determined. Chemical equilibria are determined by the changes in free energy that occur when reactants combine or move from one phase to another. The binding of small organic and inorganic molecules to biopolymers a common method of handling the binding data makes use of linear equations. Both of these equations suffer from the limitation of a relatively uncertain extrapolation and the error inherent in taking the slope of a line drawn through experimental points, which are themselves subject to a finite uncertainty.

The Measurement of Complex Formation

Publisher Summary nThis chapter discusses the measurement of complex formation. The ligand constitutes the only ionic species present, a membrane selectively permeable to ions having the same charge as the ligand can be interposed between the mixture and a solution containing a known activity of ligand alone. A membrane potential will develop that can be used to calculate the activity of the ligand in the presence of a binding body. As a practical matter, the method of equilibrium dialysis, although more widely useful than any other, suffers from a number of serious disadvantages. The presence of trace impurities, assayed as ligand, can have even more serious consequences than introduction of error in the determination of Ceq. Subtractive methods, direct measurement, electrostatic methods, etc are used for measuring complex formation.

Binding of Neutral Molecules

Publisher Summary nThis chapter describes binding of neutral molecules. The classical examples of the binding of neutral molecules by dissolved proteins are interaction with solvent and the reversion binding of oxygen, carbon monoxide, and other neutral ligands by the respiratory proteins. There is relatively little solid quantitative information available about the hydration of dissolved proteins; in fact, the concept itself is both conceptually and operationally vague. On the other hand, the reactions of respiratory proteins with neutral ligands are intimately related to interactions with the heme moiety, or with iron itself, and, thus, are a highly specialized group of interactions peculiar to the prosthetic group rather than to proteins in general. There are both advantages and disadvantages in beginning a discussion of complex formation by proteins with a review of the results obtained with serum albumins.

IX – Summary and Conclusions

Publisher Summary nThis chapter presents summary and conclusions on binding forces involved in complex formation. The most familiar multiple equilibria of proteins, shown by their hydrogen–ion titration curves, have both formal and real parallels with the multiple binding by protein of many other neutral and charged substances. The greatest differences (from H+ binding) arise with the binding of neutral hydrocarbons, where apparently only entropic forces are expressed and heats of reaction are zero or positive. The difference extends to a possible absence of stoichiometry when some (but not all) proteins bind small hydrocarbons. Globular proteins differ among themselves more with respect to their tendency to bind hydrocarbons than they do in any other easily measured property; it seems likely that this property gives both qualitative and quantitative information about the nature of their surfaces. There has been a tendency throughout this book to think of the interactions of multiple equilibria very largely in terms of hydrophobic interaction modified in some cases by electrostatic effects of smaller magnitudes and perhaps to a small extent by hydrogen bonding as well. Successful scientific exploration is characterized by a tendency to oversimplify and to over rationalize just before a sharp change of course to a new direction. The binding interactions of proteins may prove no exception to this general rule.

Protein-Protein Interaction

Publisher Summary nThis chapter discusses protein–protein interaction. The study of interactions between protein molecules is such a vast and diversified area that a comprehensive treatment of this subject will appear in another monograph in this series. There are two types of equilibria between macromolecules: (1) the interacting species are subunits which associate to form biologically active units and (2) interaction between distinct macromolecular entities occurs as a step in a biological process. There is also some indirect evidence from solution physical chemistry that some polymeric proteins owe their associative stability to hydrophobic bonding. The major problem that arises in an investigation of the formation of quarternary structure in solution, however, is the experimental recognition of alterations in the secondary and tertiary structure of the individual polypeptide chains as separate from the interaction between two such chains. Tobacco mosaic virus is a rodlike molecule consisting of 2130 protein subunits and a single RNA molecule occupying a hollow core inside the assembly of polypeptide chains. The protein component can be separated intact from the RNA and reversibly dissociated by urea, 67% acetic acid, or dilution.