Enzymes (Second Edition)

70 STRUCTURAL COMPONENTS OF ENZYMES

Figure 3.19 Examples of some common cofactors found in enzymes.

Cofactors fulfill a broad range of reactions in enzymes. One of the more common roles of enzyme cofactors is to provide a locus for oxidation/reduction (redox) chemistry at the active site. An illustrative example of this is the chemistry of flavin cofactors.

Flavins (from the Latin word flavius, meaning yellow) are bright yellow ( 450 nm) cofactors common to oxidoreductases, dehydrogenases, and electron transfer proteins. The main structural feature of the flavin cofactor is the highly conjugated isoalloxazine ring system (Figure 3.19). Oxidized flavins readily undergo reversible two-electron reduction to 1,5-dihydroflavin, and

SUMMARY 71

thus can act as electron sinks during redox reactions within the enzyme active site. For example, a number of dehydrogenases use flavin cofactors to accept two electrons during catalytic oxidation of NADH (another common enzyme cofactor; see Nicotinamide in Figure 3.19). Alternatively, flavins can undergo discrete one-electron reduction to form a semiquinone radical; this can be further reduced by a second one-electron reduction reaction to yield the fully reduced cofactor. Through this chemistry, flavin cofactors can participate in one-electron oxidations, such as those carried out during respiratory electron transfer in mitochondria. There are actually two stable forms of the flavin semiquinone that interconvert, depending on pH (Figure 3.20) The blue neutral semiquinone occurs at neutral and acidic pH, while the red anionic semiquinone occurs above pH 8.4. Both forms can be stabilized and observed in certain enzymatic reactions.

Additional chemical versatility is demonstrated by flavin cofactors in their ability to form covalent adducts with substrate during redox reactions. The oxidation of dithiols to disulfides by the active site flavin of glutathione reductase is an example of this. Here the thiolate anion adds to the C4a carbon of the isoalloxazine ring system (Figure 3.21A). Likewise, in a number of flavoenzyme oxidases, catalytic reoxidation of reduced flavin by molecular oxygen proceeds with formation of a transient C4a peroxide intermediate (Figure 3.21B).

A variety of other cofactors participate in the catalytic chemistry of the enzyme active site. Some additional examples of these are listed in Table 3.4. This list is, however, far from comprehensive; rather it gives just a hint of the breadth of structures and reactivities provided to enzymes by various cofactors. The texts by Dixon and Webb (1979) Walsh (1979), and Dugas and Penney (1981) give more comprehensive treatments of enzyme cofactors and the chemical reactions they perform.

3.8 SUMMARY

In this chapter we have seen the diversity of chemical reactivities that are imparted to enzymes by the structures of the amino acid side chains. We have described how these amino acids can be linked together to form a polypeptide chain, and how these chains fold into regular patterns of secondary and tertiary structure. The folding of an enzyme into its correct tertiary structure provides a means of establishing the binding pockets for substrate ligands and presents, within these binding pockets, the chemically reactive groups required for catalysis. The active site of the enzyme is defined by these reactive groups, and by the overall topology of the binding pocket. We have seen that the chemically reactive groups used to convert substrate to product molecules are recruited by enzymes, not only from the amino acids that make up the protein, but from cofactor molecules as well; these cofactors are critical components of the biologically active enzyme molecule. In Chapter 6, we shall see how the

ENZYMES OF COMPONENTS STRUCTURAL 72

Figure 3.20 Structures of the flavin cofactor in its various oxidation states.

structural details of the enzyme active site facilitate substrate binding and the acceleration of reaction rates, which are the hallmarks of enzymatic catalysis.

REFERENCES AND FURTHER READING

Branden, C., and Tooze, J. (1991) Introduction to Protein Structure, Garland, New York.

Chotia, C. (1975) J. Mol. Biol. 105, 1.

Copeland, R. A. (1994) Methods for Protein Analysis: A Practical Guide to L aboratory Protocols, Chapman & Hall, New York.

Creighton, T. E. (1984) Proteins, Structure and Molecular Properties, Freeman, New

York.

Davis, J. P., and Copeland, R. A. (1996) Protein engineering, in Kirk-Othmer Encyclopedia of Chemical Technology, Vol. 20, 4th ed., Wiley, New York.

Dayoff, M. O. (1978) Atlas of Protein Sequence and Structure, Vol. 5, Suppl. 3, National Biomedical Res. Foundation, Washington, D.C.

Dill, K. A., Bromberg, S., Yue, K., Fiebig, K. M., Yee, D. P., Thomas, P. D., and Chan, H. S. (1995) Protein Sci., 4, 561.

Dixon, M., and Webb, E. C. (1979) Enzymes, 3rd ed., Academic Press, New York.

Dugas, H., and Penny, C. (1981) Bioorganic Chemistry, A Chemical Approach to Enzyme Action, Springer-Verlag, New York.

Hansch, C., and Coats, E. (1970) J. Pharm. Sci. 59, 731.

Klapper, M. H. (1977) Biochem. Biophys. Res. Commun. 78, 1018.

Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105.

Pauling, L., Itano, H. A., Singer, S. J., and Wells, I. C. (1949) Science, 110, 543. Picot, D., Loll, P., and Garavito, R. M. (1994) Nature, 367, 243.

Stryer, L. (1989) Molecular Design of L ife, Freeman, New York. Walsh, C. (1979) Enzyme Reaction Mechanisms, Freeman, New York.

78 PROTEIN--LIGAND BINDING EQUILIBRIA

typical way of expressing the Gibbs free energy. This is because in general chemistry texts, the free energy is more usually presented in terms of the forward reaction, that is, in terms of the association constant:

The sign conversion between Equations 4.4 and 4.6 merely reflects the inverse relationship between equilibrium association and dissociation constants:

K

At this point the reader may wonder why biochemists choose to express affinity relationships in terms of dissociation constants rather than the more conventional association constants, as found in most chemistry and physics textbooks. The reason is that the dissociation constant has units of molarity and can thus be equated with a specific ligand concentration that leads to half-maximal saturation of the available receptor binding sites; this will become evident in Section 4.3, where we discuss equilibrium binding experiments.

4.2 THE KINETIC APPROACH TO EQUILIBRIUM

Let us begin by considering a simple case of bimolecular binding without any subsequent chemical steps. Suppose that the two molecules, R and L, bind reversibly to each other in solution to form a binary complex, RL. The equilibrium between the free components, R and L, and the binary complex, RL, will be governed by the rate of complex formation (i.e., association of the complex) and by the rate of dissociation of the formed complex. Here we will define the second-order rate constant for complex association as k and the first-order rate constant for complex dissociation as k .

The equilibrium dissociation constant for the complex is thus given by the ratio of k to k :

Figure 4.1 Time course for approach to equilibrium after mixing of a receptor and a ligand.

The data are fit by nonlinear regression to Equation 4.10, from which an estimate of the observed pseudo-first-order rate constant (k ) is obtained.

In the vast majority of cases, the strength of interaction (i.e., affinity) between the receptor and ligand is such that a large excess of ligand concentration is required to effect significant binding to the receptor. Hence, under most experimental conditions association to form the binary complex proceeds with little change in the concentration of free (i.e., unbound) ligand; thus the association reaction proceeds with pseudo-first-order kinetics:

where [RL] is the concentration of binary complex at time t, [RL] is the concentration of binary complex at equilibrium, and k is the experimentally determined value of the pseudo-first-order rate constant for approach to equilibrium. Figure 4.1 illustrates a typical kinetic progress curve for a receptor—ligand pair approaching equilibrium binding. The line drawn through the data in this figure is a nonlinear least-squares best fit to Equation 4.10, from which the researcher can obtain an estimate of k .

For reversible binding, it can be shown that the value of k is directly proportional to the concentration of ligand present as follows:

Hence, one can determine the value of k at a series of ligand concentrations from experiments such as that illustrated in Figure 4.1. A replot of k as a function of ligand concentration should then yield a linear fit with slope equal to k and y intercept equal to k (Figure 4.2). From these values, the equilibrium dissociation constant can be determined by means of Equation 4.9.