5.2: Enzymes and the Mechanisms of Enzyme Catalysis

Studies by George W. Beadle and Edward L. Tatum correlating mutations with enzyme deficiencies in Neurospora crassa (bread mold) and Drosophila melanogaster led them to propose the one-gene/one-enzyme hypothesis in 1941. By 1958, they shared the Nobel Prize in Physiology or Medicine for this work. Their original hypothesis had already morphed twice—first into the one-gene/one-protein, and then into the one-gene/one-polypeptide hypothesis…, helping to launch the age of molecular biology. The subsequent discovery of RNA catalysts came as quite a surprise! The revelation of RNA catalysts, dubbed ribozymes, earned Sidney Altman and Thomas Cech a Nobel Prize in Chemistry in 1989. Ribozymes are now known to catalyze RNA splicing (the removal of unwanted regions of a precursor RNA). They also catalyze a step in the protein synthesis (translation) by ribosomes. In fact, almost no biochemical reactions or pathways exist that are not directly the result of enzyme catalysis, from the digestion of nutrients in your mouth, stomach, and small intestines to pretty much every chemical reaction inside your cells (check out Kornberg A. 1989. Never a Dull Enzyme. Ann. Rev. Biochem. 58:1-30). The focus in this chapter is on the long history of protein enzyme catalysis. But as you study, you may recognize that the mechanisms of enzyme catalysis described here involve common essential features seen in all biocatalysts.

Most enzymes are soluble inside or outside cells, while a few are part of membranes or other cellular structures. In all cases, they bind to soluble substrates (the reactants in enzyme-catalyzed reactions). The large size and exquisite diversity of protein structures make enzymes highly specific catalysts. As already noted, the specificity of an enzyme results from the shape of its active site, which is dependent on the 3D arrangement of amino acids in and around the region. The substrates of a catalyzed biochemical reaction are bound to and held in place on the enzyme while rapid bond rearrangements take place. Their flexibility allows enzymes to change in shape at the active site during catalysis. In addition, this flexibility enables small metabolites in cells to interact with an enzyme, change its shape and thereby changing its catalytic rate. The latter phenomenon enables allosteric regulation, allowing cells to control the rates and even the direction of many biochemical reactions and pathways. As we will see, enzymes may be bound to prosthetic groups or ions, which contribute to the shape and activity of the enzyme.

Any understanding the mechanism of catalysis must also include knowledge about the energetics of catalyzed reactions. We’ll see that enzymes lower the activation energy of a chemical reaction, and that activation energy is an inherent energy barrier to the reaction. Finally, we look at the energetics of enzyme action.

In sum, we will describe the action of biological catalysis in terms biochemical pathways and the structural features of enzymes (e.g., active-site and overall conformation and the affinities of an enzyme for its substrates), as well as free energy changes that occur during catalysis. Of course, structural and energy considerations of enzyme catalysis are related.

145 Enzymes vs Other Catalysts

5.2.1. Structural Considerations of Catalysis

From a chemistry course, you may recall that the rate of an uncatalyzed reaction is dependent on the concentration of the reactants in solution. This is the law of mass action, recognized in the nineteenth century. Look at this simple reaction:

\(A + B \rightleftarrow C + D\)

The law of mass action makes two key assumptions:

1. At any given time following the start of the reaction, the rate of product formation is proportional to the concentrations of the reactants and products ([A], [B], [C], and [D] in this case).
2. Chemical reactions in the laboratory (i.e., a “closed system”) eventually reach equilibrium, at which point the net rate of formation of reaction products is zero. (in other words, the forward and reverse reactions occur at the same rate.)

CHALLENGE

Imagine and graph the expected rate of our chemical reaction over time, plotting time on the X axis and concentration of product (say, [C]) measured at different times.

There are no products (i.e., C and D) at the start of the reaction written above. As there are no products yet, the reaction rate should be directly proportional only to the concentration of the reactants. The law of mass action predicts that the chemical reaction above will occur faster when A and B are first mixed—that is, when A and B at their higher concentrations. This is because there are more reactant molecules in solution and a greater likelihood that they will collide in an orientation that allows the bond rearrangements necessary for the reaction to occur.

Of course, reactant concentrations decline as products accumulate over time, so the rate of formation of C and D must slow down as reactant levels diminish. At some point, as products accumulate, they should also influence the rate of their own production. Remember, all chemical reactions are inherently reversible So, rising levels of products will begin to push the reaction in reverse to form A and B, slowing down the net accumulation of C and D.

The chemical rate equations that you may recall from a chemistry course in fact treat all chemical reactions as reversible (elsewhere we’ll address the concept that some reactions in cells are biologically irreversible!). Chemical rate equations enable the determination of reaction rates for our sample reaction. Here is an equation for the rate of formation of the products C & D:

\(\text{rate of formation of C and D} = k_1[A][B] - k_{-1}[C][D]\)

In this equation, \(k_1[A][B]\) is the rate of the forward reaction and \(k_{-1}[C][D]\) is the rate of the reverse reaction. This equation recognizes that the reaction is reversible. The equation states that the net reaction rate is equal to the rate of the forward reaction \(k_1[A][B]\) minus the rate of the back reaction \(k_{-1}[C][D]\). The equation is valid (applicable) at any time during the reaction. \(k_1\) and \(k_{-1}\) are rate constants for the forward and reverse reactions, respectively.

CHALLENGE

Using the equation for the rate of formation of C and D, can you determine what the rate will be at the end of the reaction? Answer this question arithmetically and include a text explanation.

So how do catalysts work? Catalysts increase chemical reaction rates by bringing reactants (now called substrates) together more rapidly than they would encounter each other based just on random molecular motion in solution. This is possible because catalysts have an affinity for their substrates. In the case of inorganic catalysts, relatively weak, generic forces account for the affinity of reactants and inorganic catalysts. Thus, a metallic catalyst (e.g., silver or platinum) attracts molecules with an appropriate (usually a charge) configuration. If the attraction (affinity) is sufficient, the metal will hold reactants in place long enough to catalyze the bond rearrangements of a chemical reaction. In contrast to inorganic catalysts, enzymes have evolved highly specific shapes with physical-chemical properties. As a result, typical enzymes only attract substrates for specific reactions, and with high affinities. The lock and-key mechanism was the first explanation for enzyme-substrate specificity (Figure 5.1).

Figure 5.1: In the lock-and-key model, reactants i.e., substrates (S) are ‘keys’ that fit in enzyme (E) ‘locks’: (1, left), E and S attract each other; (2, middle) S enters the catalytic site on E like a key in a lock, forming an E-S complex (middle); (3, right) catalysis occurs, E and P separate.

The active site of an enzyme has an exquisite, selective affinity for its substrate(s). This affinity is many times greater than those of inorganic catalysts for generic reactants. The result is that enzymes are more efficient and faster than inorganic catalysts. According to this model, the affinity of an enzyme for a particular substrate engages the substrate “key” in the tumblers (i.e., in the active site) of the enzyme’s “lock” Thus engaged, the substrate(s) would undergo the bond rearrangements specific for the catalyzed reaction to generate products and to regenerate an unchanged enzyme.

However, X-ray crystallography of enzyme-substrate interactions revealed that the active sites of enzymes change shape during catalysis. This allosteric change suggested the induced-fit mechanism of enzyme action (modeled in Figure 5.2).

Figure 5.2: In the induced-fit model, E and S attract each, other initially forming a short-lived ES*, inducing an allosteric (shape) change leading to a tight fit of S in the catalytic site of E to form an E-S complex. Catalysis occurs and E and P separate.

In this model, enzyme-substrate affinity causes the substrate to bind to the enzyme surface. Once bound, the enzyme undergoes an allosteric change, drawing the substrate(s) more tightly into the active site and catalyzing the reaction. Of course, after the reaction products detach from the enzyme, it returns to its original shape.

146-2 Induced-Fit Mechanism of Enzyme Action

CHALLENGE

Note that when an enzyme (or any catalyst) catalyzes a biochemical reaction in a closed system, it still reaches equilibrium, despite the higher rate of the catalyzed reaction. What does this tell you about the energetics of catalyzed reactions in closed systems? And why do biochemical pathways never reach equilibrium?

5.2.2. Energetic Considerations of Catalysis

Consider the random motion of substrates in solution, which only occasionally encounter one another. They even more rarely bump into one another in just the right orientation to cause a reaction. This explains why adding more reactants or increasing the temperature of a reaction can speed it up: it increases the number of random as well as productive molecular collisions. Unlike molecules and reactions in a test tube, living organisms do not have these options for increasing reaction rates. But they do have enzymes! All catalysts work by lowering the activation energy (\(E_a\)) for a reaction, thereby increasing the rate of the reaction. Activation energy is essentially a barrier that makes it difficult for interacting substrates to come together to actually undergo a biochemical reaction.

Inorganic catalytic surfaces attract reactants where catalysis can occur. The attractions are weak compared to those of enzymes and their substrates. An enzyme’s active site very strongly attracts otherwise randomly distributed substrates, making enzyme catalysis faster than inorganic catalysis. Again, cells cannot use inorganic catalysts, most of which are insoluble and would attract reactants indiscriminately—not a good way for cells to control metabolism! The advent of enzymes with their specificity and high rate of catalysis was a key event in chemical evolution and was required for the origins of life. As we saw, allosteric change during the “induction of fit” enables specific catalysis. In fact, a catalyzed reaction will be faster than the same reaction catalyzed by a piece of metal, and of course much faster (millions of times faster!) than the uncatalyzed reaction. The energetics of catalysis helps to explain why. Let’s look at the energetics of a simple reaction in which A and B are converted to C and D (Figure 5.3).

Figure 5.3: This graph follows the free energy change over time as reactants A and B react. The activation energy peak in the middle of the curve defines that free energy barrier which A and B must overcome before much C and D can be made. The barrier is due mainly to thermal motion of A and B molecules that rarely encounter each other in relatively dilute solutions. Enzymes are even more efficient that inorganic catalysts in lowering the activation-energy barrier to a reaction.

Conducted in a closed system, enzyme-catalyzed reactions rapidly reach equilibrium. Like all catalysts, enzymes are not consumed by the reactions they catalyze, nor do they alter equilibrium concentrations of reactants and products of these reactions. One estimate is that as few as 4,000 biochemical reactions are catalyzed in a given cell at a given time. Another estimate is that there are 20,000-25,000 genes in the human genome. Many of these encode enzymes. What are these genes and their protein products good for if so few biochemical reactions are needed by an average cell? Is this in fact an underestimate?

147 Enzyme Activation Energy

CHALLENGE

What do you make of what seems to be an inherent conflict between the estimates of the number of genes and the number of enzymecatalyzed reactions in cells?