CHEM 440 - Enzymology: An introduction

BIOCHEMISTRY TOPICS

Enzymology: An introduction

General properties of enzymes. Enzyme classification. Enzyme specificity: Enzyme-substrate complementarity. Cofactors, coenzymes, and prosthetic groups. Activation energy and reaction coordinate diagrams. Transition state theory; reduction of ΔG‡ by catalysts. Catalytic mechanisms: General acid/general base catalysis, covalent catalysis, metal ion catalysts, proximity and orientation effects. Enzymes preferentially bind the reaction transition state.

Enzymes: Basic terminology and concepts

A catalyst is a substance that speeds up the rate of a chemical reaction without itself being consumed or generated by the reaction. An enzyme is a protein that acts as a catalyst. The term substrate refers in enzymology to a reactant in an enzyme-catalyzed reaction. The rate enhancement achievable by an enzyme − which can speed up a reaction by many orders of magnitude compared to its non-enzymatic rate - is accounted for by the direct interaction at the molecular level between enzyme and substrate. This interaction occurs, first forming an enzyme-substrate (ES) complex, in a part of the protein molecule (proteins are typically much larger than their substrates) called the active site. The features of active sites are described further below. Another characteristic of enzymes is their substrate specificity. In many instances enzymes show an exquisite ability to discriminate between closely related molecules. Through methods for structure determination, the ES complex or its analog can be directly observed. Enzymes are involved in virtually every biological process. They are primarily and historically connected to metabolic reactions, but enzymes also participate in processes of regulation and energy transduction.

Enzyme classification

Enzymes are classified according to the type of reactions they catalyze.

Oxidoreductases (redox reactions) Example: lactate dehydrogenase [EC 1.1.1.27]
Transferases (transfer of functional groups) Example: aspartate transcarbamoylase (ATCase) [EC 2.1.3.2]
Hydrolases (hydrolysis reactions) Example: RNase A [EC 3.1.27.5]; lysozyme [EC 3.2.1.17]
Lyases (group elimination to form double bonds) Example: carbonic anhydrase [EC 4.2.1.1]
Isomerases (isomerization) Example: triose phosphate isomerase (TIM) [EC 5.3.1.1]
Ligases (joining molecules with ATP or other NTP hydrolysis) Example: pyruvate carboxylase [EC 6.4.1.1]

Chemical thermodynamics, kinetics, and enzymology

In applying the principles of chemical thermodynamics to biological catalysis, we find that under the typical physiological conditions of constant temperature and pressure, the Gibbs free energy function (G) is most useful. The free energy change, ΔG, for a process (e.g. chemical reaction) determines if that process is spontaneous under specified conditions. Equilibrium conditions are governed by the standard free energy change ΔG°. Note that in biochemistry, ΔG°′, the change in biochemical standard free energy, is used. Recall the following key points about free energy:

1. If a process, occurring under conditions of constant temperature and pressure, has a negative Gibbs free energy change (i.e. ΔG < 0), then that process is spontaneous, in a thermodynamic sense (but see 5, below). If a process is observed to occur spontaneously, ΔG must be less than zero for that process.

2. The equilibrium condition is ΔG = 0. This is equivalent to a condition for which the free energy G, as a function of product and reactant concentrations, is at a minimum. The equilibrium is dynamic, but no net change in composition occurs because G has attained its minimum value.

3. If ΔG > 0, for a process, it cannot occur spontaneously. The process must be coupled to another process for which ΔG < 0 and of greater magnitude, so that the net ΔG for the sum of the two processes is negative.

4. The relationship between free energy and the equilibrium constant is defined by the equation ΔG°′ = −RT ln K′, where ΔG°′ is the biochemical standard free energy change for the reaction, and K′ is the equilibrium constant.

5. Even if ΔG < 0 for a process, this provides no information about the rate of that process. Rates of chemical reactions can be understood in terms of transition state theory. Of course, in biochemistry, the presence of enzymes insures that the reactions catalyzed by them will proceed more rapidly toward equilibrium.

In assessing ΔG´ for in vivo processes, such as those associated with metabolic reactions, we may suffer the limitation of inaccurately known concentrations of reactant and product species. In cases where good data on in vivo concentrations of metabolites is unavailable, ΔG°´ values are used for comparative purposes.

By analogy to the methods of equilibrium thermodynamics (Big K), a kinetic rate constant (little k) can be related to the theoretical free energy required to reach the transition state (ΔG^‡). This approach to kinetics is referred to as transition state theory. Bearing in mind the distinction between thermodynamics and kinetics, we present the following key principle of enzymatic catalysis

While enzymes never(!) alter the ΔG of reaction, they enhance the rate of approach to equilibrium by selectively lowering ΔG^‡ for the reaction after forming an ES complex. This transition state stabilization, or preferential binding in a transition state (ES^‡) complex, is an important mechanistic principle.

In studying enzymes and their complexes with substrates or inhibitors, we aim to learn how the enzyme active site is adapted to bind cognate substrates and selectively lower the energy of the transition state(s) for the reaction.

Transition state theory

We can apply the formalism of thermodynamics to derive a general theoretical expression for the rate of a chemical reaction that quantitatively relates the velocity of a reaction to the free energy of activation, ΔG^‡. This in turn, will allow us to quantify the rate enhancement that an enzyme can achieve by reducing the value of ΔG^‡ by a certain amount. By assuming the reactant is in equilibrium with the reaction transition state, an equation is derived that shows that the velocity (rate) of reaction is proportional to the concentration of the reactant and the exponential of the ratio (−ΔG^‡/RT). Using the appropriate form of this relation, it can be shown that at 25°C an enzyme can achieve a 10-fold rate enhancement for each 5.71 kJ/mol (1.36 kcal/mol) lowering of ΔG^‡.

Enzyme mechanisms and the chemistry of catalysis

A reaction catalyzed by an enzyme may entail a sequence of several distinct elementary steps, and thus a number of transition states. The study of enzymes is greatly concerned with a detailed description of this sequence of elementary steps, or enzyme mechanism. Proteins make good enzymes because of their structural and chemical diversity. Furthermore, as we'll soon see, the chemical repertoire of enzymes is greatly extended by non-protein cofactors. Despite the great variety of players in mechanistic models explaining how enzymes work, some common themes emerge from the bulk of studies of many different enzymes. Looked at in reverse, as a unifying approach in biochemistry, we can attempt to apply a few mechanistic principles to the great variety of enzymes and the reactions they catalyze. This is also where familiarity with the mechanisms in organic chemistry pays real dividends, since (as one respected enzymologist put it - see Ref. 4) enzymes are not different, just better. The reactivity of carbonyl compounds and carboxylic acid derivatives underlies the logic of many metabolic reactions. In terms of chemical species that mediate catalysis, the most common mechanistic types are general acid/general base catalysis, covalent catalysis, and metal ion catalysis.

The chemical diversity of the twenty amino acids makes proteins well-suited to catalyze reactions via a number of common mechanisms of the first two types. Many reactions in organic chemistry proceed via general acid or general base mechanisms, or involve nucleophilic attack, which figures prominently in covalent catalysis. Groups such as -COOH and ImH⁺ (protonated imidazole) can act as general acids (or proton donors), while groups such as -COO⁻, Im (neutral imidazole), and -NH₂ can act as general bases (proton acceptors) in catalytic mechanisms. The side chains of proteins include groups that can act as nucleophiles such as the -OH and -SH groups of Ser and Cys, respectively.

Cofactors

There are some catalytic chemistries for which protein groups alone are not so well-suited, for example electrophilic catalysis. To extend their catalytic capabilities, enzymes often make use of accessories, generally termed cofactors. Cofactors can be defined in general as "accessories" upon which the catalytic activity of many enzymes depend. These cofactors are further subdivided into two groups: metals and "small" organic molecules. The term coenzyme is typically applied to the latter, and a coenzyme that is very tightly bound (often covalently linked) - and therefore not exchanged by the enzyme is referred to as a prosthetic group. The roles that cofactors play in enzyme activity are varied. In some cases, they are clearly catalytic in nature, helping enzymes perform some chemistry that proteins in general are not particularly adept at. In other cases, the coenzymes act as modular carriers of chemical species such as electrons, one-carbon units, or acyl groups. In this role, the term cosubstrate is perhaps more apt, but their modular nature - being exchanged as part of catalytic turnover, recycled and used by a range of enzymes - and their relationship to vitamins (from which they are derived) distinguishes them from typical substrates.

Metal ion cofactors often perform a distinct catalytic role. The Zn²⁺ in carbonic anhydrase provides a clear example of metal ion catalysis. Cosubstrates and prosthetic groups can act as carriers of electrons (NADH, FADH), or chemical groups Coenzyme A is a carrier of acyl groups). The versatile coenzyme pyridoxal phosphate (PLP) can act as an acceptor of amino groups. These are just a sampling of types and roles of cofactors encountered in enzymes.

Enzyme active sites and enzyme-substrate (ES) complexes

The active site is a three-dimensional cleft or crevice or "pocket" that forms by the juxtaposition of different residues in the tertiary structure of the enzyme. In view of the difference between primary and tertiary structure, it should come as no surprise that sometimes residues quite far separated in the sequence contribute to the formation of an enzyme active site. Enzymes accomplish great catalytic feats, not by magic, but by direct physical interaction between enzyme and substrate. Even before the advent of methods to directly determine structures of enzymes and their complexes, several lines of evidence pointed to the formation of enzyme-substrate complexes. First, enzymes were long known to show saturation kinetics. Reaction velocity as a function of substrate concentration reaches a maximum, suggesting a saturation of catalytic sites on the enzyme where the enzyme and substrate interact. Spectroscopic methods such as UV-visible or fluorescence spectroscopy have been used to infer change to enzymes upon addition of substrates. X-ray crystallography of enzyme-substrate or enzyme-substrate analog complexes has now yielded many, many examples of how enzymes recognize and bind their substrates, in explicit structural detail.

Recognizing that the majority of enzymatic reactions involve more than one substrate (reactant) leads us to another important mechanistic principle, which is to consider proximity and orientation effects. A bisubstrate reaction may occur in several different patterns as we shall see, but if an enzyme catalyzes a reaction involving substrates A and B, a ternary enzyme-substrate complex (here we could denote it as EAB) may well form. In this context, we can see that an important way in which an enzyme can be conceived to enhance the reaction A + B → products is simply by bringing the substrates together within the friendly confines of the active site. This can be understood as vastly increasing the effective concentration of the reactants, which we know from chemical kinetics increases reaction rates. Furthermore, if A and B are brought together in close proximity, it would seem crucial in most instances for that to be done in such a way so as to at least allow the best orientation of A and B for the reaction to take place. The directionality of nucleophilic attack in the geometry of the S_N2 reaction from organic chemistry well illustrates the importance of orientation. What an enzyme can do exceedingly well is not only bind substrates together within the active site, but also in a precise orientation favorable to the reaction being catalyzed.

Binding energy

In order to understand preferential binding of the transition state, it is helpful to consider the concept of binding energy, the energy change in the formation of the ES complex, and then to ask the question: Does the binding energy of a substrate represent the maximum achievable affinity of enzyme for substrate, or could an enzyme utilize a fraction of binding energy in order to be more adapted to binding a transition state species?

The lysozyme example is a particularly good illustration of binding energy. Its rather large substrate makes many energetically favorable contacts, contributing to strong binding. However, lysozyme does not bind its substrate as tightly as possible - its role is to hydrolyze its substrate, not bind it as tightly as possible. Some of the contacts the enzyme makes help distort part of the substrate so that it more resembles the reaction transition state. This is another way of looking at the principle regarding selective lowering of the reaction transition state energy expressed above. lysozyme binds its substrate well, but binds the transition state even better, a key distinction that makes the catalytic difference. Some of the potential binding energy in forming the enzyme-substrate complex is sacrificed, in a sense, in order to more effectively bind a higher energy form of the substrate.

Having made our introductory acquaintance with enzymes, we follow up with case studies of enzymes with particular emphasis on how they achieve such catalytic power, employing mechanisms that fit within our framework and also the modes by which substrate specificity is achieved.

Catalytic power and specificity are both related to shape (or steric) complementarity and electrostatic complementarity. Complementarity to the transition state lowers the activation energy for a reaction, and complementarity to substrate has much to do with specificity. Because proteins can adopt a variety of shapes and create precisely adapted electrostatic environments, a process of natural selection can act, resulting in the evolution of proteins that are highly efficient enzymes.