FUNCTIONAL ASPECTS OF ENZYMES

IV. FUNCTIONAL ASPECTS OF ENZYMES

Enzymes have been defined as powerful catalysts. The catalytic efficiency of enzymatic reactions approaches 10 8 –10 11 . Catalytic factors are obtained by dividing the rate of a reaction catalyzed by an enzyme by the rate of the same reaction in the absence of the enzyme. In enzyme kinetics, V max /K m ratios are commonly used to assess the catalytic power of enzymes. Calculations have shown that one enzyme molecule can transform as

many as 10 6 substrate molecules per minute. These speeds are achieved at low temperature, under mild pH conditions, and in a solvent that is inexpensive, safe, and abundant, namely, water. Another advantage of enzymes is the wide variety in nature that one can select from. A similar smorgasbord of chemical catalysts would not be easy to compile. As discussed under nomenclature, there is a sub-subclass of enzyme for every type of chemi- cal reaction conceivable. For example, not only can enzymes participate in isomerization reactions, but they can do so in racemic and epimeric modes, facilitate cis-trans isomeriza- tion, or cause intramolecular oxidation/reduction or group transfers. Further, among the racemases and epimerases there are specific enzymes that act only on amino acids and their derivatives, hydroxy acids and their derivatives, carbohydrates and their derivatives, or other compounds. One of the exciting prospects of enzyme technology is the creation of ‘‘synzymes’’—synthetic enzymes that can catalyze reactions not necessarily observed in nature. These synzymes can be viewed as enzyme analogs. Enzymes also promote ‘‘clean’’ reactions, that is, the tendency to form undesirable byproducts is minimized. Chemical catalysts, on the other hand, have a propensity to produce undesirable byprod- ucts. This dedication of enzymes to act on very few substrates to produce specific products is what is termed substrate specificity, but substrate specificity is not absolute and must

be addressed in the context of reaction conditions such as solvent, temperature, and pH. Industrially important enzymes are not normally used to convert their physiological sub- strates. For example, glucose isomerase used in the production of high-fructose corn syrup actually preferentially acts on xylose, its physiological substrate (Bucke, 1977). The speci- ficity of enzymes can also be extended to stereospecificity, or the ability to distinguish between chiral carbon atoms. Enzymes have one other advantage in that they can be syn- thesized or degraded in rather short order, providing an attractive method for gross control over reactions. Further, binding of small molecules can modify enzyme activities, thereby offering finer control elements in reactions.

Thermodynamic laws, applicable to all reactions, do not make exceptions for en- zymes. A catalyst, whether chemical or biochemical, speeds up the rate of reaction but does not alter the equilibrium constant. In order for a bond to be made, broken, or trans- formed, energy is required. Energy is consumed during the formation of bonds and re- leased during the breaking of the bonds. Energy needed for the bond brakeage has to be obtained from molecular collisions. This energy will be stored until a transition state is formed, and the energy input is called free energy of activation. After this free energy activation is achieved, the reaction proceeds spontaneously, leading to bond breakage.

Enzymes, being catalysts, provide new reaction pathways in which (1) and the slow- est step (called the rate-limiting or rate-determining step) has a lower free energy of activa- tion than the rate-determining step of the uncatalyzed reaction, and (2) all transition state energies in the catalyzed pathway are lower than the highest one of the uncatalyzed path- way. In an enzyme-catalyzed reaction a third possibility is the creation of the environment in which the free energy of the product is lowered. The formation of transition complexes between the enzyme and the substrate takes 10 ⫺9 –10 ⫺6 s, and as long as fresh substrate is available the concentration of free enzyme can be maintained in a dynamic equilibrium.

Rate constants are very sensitive to the free energy of binding of substrate. A change in only 2 kcal/mol between two competing substrates will result in a 97 : 3 ratio between their reaction rates. Enzymes do not alter the overall free energy of the reaction, but only decrease the free energy of the transition state complex. Jencks (1975) demonstrated that the slowness of non–enzyme-catalyzed and uncatalyzed reactions in free solution is due to a loss of transitional and rotational entropy resulting from the formation of the transition state complex. This explanation, however, does not apply to intramolecular reactions and enzyme-catalyzed reactions because of the formation of enzyme–substrate complexes. Further proof of this explanation was provided by showing that substrate concentration of 10 M would suffice for enzyme-catalyzed reactions, whereas for the same reaction to

proceed in free solution, reactant concentration of 10 7 –10 9 M would be necessary. All known enzymes are proteins. Proteins are polymeric entities that exhibit struc- tural hierarchy. The monomeric units are amino acids joined together by amide linkages or peptide bonds. There are 20 amino acids in nature from which enzyme molecule can

be built. Sequence of amino acids can be varied to obtained differences in proteins. The long chain of amino acids does not remain extended but rather takes up a definite three- dimensional structure, which is further stabilized by intramolecular crosslinks and interac- tions. For example, two cysteine residues can combine to form a disulfide bond, and often this facilitates the formation of ‘‘loops.’’ Similarly, hydrogen bonds, hydrophobic interac- tions, and ionic bonds all contribute to holding the coil in place. In some enzymes, poly- peptide chains may be linked together in what are called quaternary, or subunit, structure.

Enzymes are specific, and even small changes in the structure of a substrate molecule lead to the inability of the enzyme to convert the compound to products. Therefore, it was suggested that the binding of an enzyme to its substrate is analogous to a lock and key, with the key fitting only one lock. The area where the substrate is bound and transformed is called the active site, and specific residues of amino acids in the active site region are collectively called the active center. The size of the active site is not set and is perhaps dependent on the functional size of the substrate molecule. For example, degradation of

a polymer like amylose may require a larger active site than the hydrolysis of maltose to glucose. The groups involved in the active center are generally not sequentially proximal to one another, i.e., they are not near neighbors in terms of the primary structure of the protein. The highest order in structure imposed by secondary, but more often tertiary and quaternary, structure bring the group in the active center into spatial proximity. Therefore, in an enzyme the majority of the amino acid residues are involved in keeping the few necessary residues in close spatial proximity. If this spatial orientation is distorted by such factors as heat, pH, or solvent effects, the catalyst nature of the enzyme will be diminished or even lost. Such distortion can be very minute, say 0.1 A ˚ (100 picometers).

The catalytic efficiency of an enzyme may result from (1) proximity and orientation effects, (2) catalysis by distortion, (3) catalysis by acid–base reactions, and (4) nucleo- The catalytic efficiency of an enzyme may result from (1) proximity and orientation effects, (2) catalysis by distortion, (3) catalysis by acid–base reactions, and (4) nucleo-

10 4 enhancement of reaction rates in enzymes-catalyzed biomolecular reactions and 1 ⫻

10 22 for enzyme-catalyzed intermolecular reactions based on 10 ⫺3 M concentration of re- actants (Koshland, 1960). Proximity alone is not adequate to explain catalytic efficiencies; anchoring a substrate to the enzyme is another critical process. Such a binding process has to decrease the entropy. The closer the ground state resembles the transition state, the more positive the entropy and the faster will be the reaction rate (Bender et al., 1964).

The second factor, catalysis by distortion, is an elegant theory postulated to play an important role in the action of such enzymes as lysozyme, trypsin, and chymotrypsin. This hypothesis is not verifiable due to experimental limitations. It suggests that the associ- ation of enzyme and substrate places a strain on the electronic structure of the substrate.

The third factor is catalysis by acid–base reactions. Acids are defined as substances that donate proteins, while bases are proton acceptors. Many amino acids in protein chains act as acids or bases. Some of these side chains and residues are α-carboxyl groups of the C-terminus peptide, carboxyl groups of the N-terminus and lysyl residues, respectively. The sulfhydryl group of cysteine, the phenolic hydroxyl of tyrosine, and the guanidino group of arginine all act as general bases when they take up protons but also have the ability to give up protons.

A fourth mechanism postulated to operate in enzyme catalysis is dependent on the nucleophilic and electrophilic properties of the amino acids residues in the enzyme mole- cules. A nucleophile is a group that has a strong tendency to donate a pair of electrons. If the rate of the reaction depends in whole or in part on a step that involves donation of an electron pair from the catalyst to the substrate, it is termed a nucleophilic catalysis. Conversely, if the reaction rate is dependent in whole or in part on a step involving accep- tance of an electron pair from the substrate by the end product, it is termed an electrophilic catalysis.

All these briefly discussed mechanisms are only postulations, and often more than one mechanism is operational in catalysis by a single enzyme. Therefore, a thorough un- derstanding of the mechanism of catalysis may not be as important to the successful appli- cation of enzyme in food processing as it is to the use of the other food additives.