Enzymes

Enzymes in biochemistry are regulatory proteins that assist in many of the reactions and processes in living cells. The DNA of a cell contains the master plan for the creation of the enzymes that are needed for life processes, and the enzymes assist in the replication of DNA and in fact help with making other copies of themselves and other proteins. One perspective on enzymes is that they facilitate many chemical reactions that otherwise would be too slow to sustain living systems. Examples have been found where an enzyme increased the rate of a biochemical reaction by more than a billion-fold! They may have binding sites which hold two or more other molecules to facilitate their binding to one another and also a binding site for ATP to provide the energy necessary for accomplishing the bonding.

This representation of enzyme action is patterned after Karp. It pictures the action of the enzyme hexokinase in the first step of the glycolysis pathway for the breakdown of glucose. The course of the reaction is plotted in terms of the Gibbs free energy G and it can be seen that this reaction proceeds in the forward direction since the final free energy is 4 kcal/mole lower than the initial state. However, the course of such reactions require energy to break and/or make chemical bonds, so energy is typically required as an activation energy E_A to produce the transition state between the initial and final states.

The remarkable protein enzymes such as hexokinase are made only in living cells and are very specific to the reactions which they catalyze. In this example the enzyme action facilitates the formation of the transition state and lowers the amount of energy required to reach this state.

A brief summary of the properties of such enzymes:

• Enzymes are typically required only in small amounts.
• They are not altered irreversibly during the course of the reaction, so can be used repeatedly.
• They have no effect on the thermodynamics of the reaction. They do not supply energy for the reaction, and therefore do not determine whether a reaction is favorable or unfavorable.
• They accomplish their function at the mild temperature and pH of living cells.
• They can be regulated to meet the needs of the cell at a particular time.

Enzymes frequently require helper molecules called coenzymes to facilitate their work.

Enzymes Facilitate Biochemical Reactions

Enzymes are regulatory proteins that assist in many of the reactions and processes in living cells.

This perspective of the role of an enzyme is patterned after Karp. It pictures the action of the enzyme pyruvate kinase in the last step of glycolysis which produces 2 pyruvates. It also produces two units of ATP for energy. The pyruvate can be used as input to the TCA cycle and finally produces more ATP energy via the electron transport chain and oxidative phosphoryllation.

With the aid of these enzymes, biochemical reactions occur at incredible speed and provide the necessary biological molecules at a rate which makes life processes possible. One of the classic examples of enzyme action is that of OMP decarboxylase, an enzyme that catalyzes the production of pyrimidine nucleotides. Without the enzyme action, it would take 78 million years to convert half the reactants to product. The catalytic action of OMP decarboxylase accelerates the reaction by a factor of 1.4 x 10¹⁷ so that it is accomplished in about 20 milliseconds!

A crucial enzyme for the respiratory process is the enzyme carbonic anhydrase which manages the conversion of carbon dioxide and water to bicarbonate for the transport of CO₂ to the lungs in the bicarbonate form to accomplish the necessary buffering to keep the body's pH in the narrow required range. This enzyme is one of the fastest known, catalyzing up to a million reactions per second.

How is such a great increase in speed accomplished with the use of enzymes? One part of the answer is addressed in the plot of the free energy G over the course of a reaction. The enzyme catalysis allows the reaction to be accomplished with a lower activation energy and therefore a larger fraction of the reactant molecules will have a kinetic energy higher than the transition state. A major factor in determining speed is the formation of the enzyme-substrate complexes as illustrated above. The complementary shapes of the active sites of catalysts make it much more probable that the reactant molecules will reach the appropriate positions and orientations to permit the reaction to occur. Non-catalyzed reactions depend upon collisions between high speed molecules in random orientations.

In addition to the complementary shapes and orientations of the active regions of enzymes, other characteristics can contribute to the catalysis:

• Distribution of electrons in the side chains of the enzyme can facilitate the interaction.
• Introducing some strain in the substrate to modify its geometry can contribute to some interactions.
• Temporary covalent enzyme-substrate linkages
• Enzymes contain amino acids which can donate or accept protons, altering the substrate to make it more reactive.

Regulation of Enzyme Reactions

The amazing speed of enzyme-catalyzed reactions allows the necessary biochemical reactions to operate at the "speed of life", to quote Ahearn. But such reactions must not be allowed to operate at full speed continuously but must respond to the level of need in the varying conditions of living cells.

Enzyme-catalyzed reactions can be described in terms of the rate or velocity of product formation as a function of the substrate concentration supplied.

Using the notation of Karp, the variable V represents the "velocity" of the reaction, defined as the number of moles of product formed per second. Vmax is then the maximal rate of production as the amount of substrate [S] supplied approaches saturation. Also used is the "turnover number", the maximum number of molecules of substrate that can be turned into product by one molecule of enzyme per second.

The turnover number is also called the catalytic constant, k_cat. While typical turnover numbers may be in the range 1 to 1000, the remarkable enzyme carbonic anhydrase can reach 10⁶!

Another term used in the description of the catalysis is the value of the sustrate concentration [S] which is associated with a velocity V of half the maximum V_max/2. This value is labeled K_M and is called the Michaelis constant. It can be used in a relationship for the reaction velocity V called the Michaelis-Menten equation, which can be used to produce the descriptive graphic above.

Another way used to display the enzyme reaction which has some advantages is the Lineweaver-Burk plot. It plots the reciprocal 1/V of the velocity versus the reciprocal of the substrate concentration 1/[S]. The value of Vmax may be found from the intercept on the vertical axis and the value of KM may be obtained from the intercept of the horizontal axis. This plot provides an advantage if a few experimental values are collected since values along a straight line may be easily extrapolated to obtain a value for KM.

The above plots may be used to evaluate the effects of enzyme inhibitors which are used to regulate the output of the product to appropriate levels to meet the needs of a cell. Enzyme inhibitors generally take the form of molecules which bind to the enzyme to decrease its output. Enzyme inhibitors may be manufactured as drugs, antibiotics, or pesticides since they can be used to control undesirable biochemical processes.

Competitive inhibitors are molecules that bind loosely to the enzyme and are therefore reversible, but act to diminish the activity of the enzyme by competing for the active site of the enzyme. Active sites on enzymes typically have some form of complementary geometry that accommodates the substrate, so competitive inhibitors may share some of those geometric features so that the can compete for binding to the enzyme. Such competitive inhibition is the basis for the design of many common drugs.

Noncompetitive inhibitors are reversible inhibitors that bind to a different site on the enzyme and therefore do not compete for binding in the normal active site. In this case the amount of inhibition just depends upon the concentration of the inhibitor and increasing the amount of the substrate will not overcome it. As shown in the plots below, noncompetitive inhibition will lower the achievable V_max. Competitive inhibition permits the achievement of a V_max comparable to the uninhibited V_max, but will require a larger substrate concentration [S].

Irreversible inhibitors are agents that bind tightly to an enzyme, often by forming covalent bonds to one of the amino acids contained in the enzyme. Such inhibitors may completely halt a biochemical process and can form potent drugs or poisons. Some nerve gases and organophosphate pesticides act as irreversible inhibitors of acetylcholinesterase. That enzyme normally breaks down the acetylcholine which stimulates muscle contraction. With that enzyme blocked, the acetylcholine continues to stimulate the muscle so that it remains in a state of permanent contraction. The antibiotic penicillin acts as an irreversible inhibitor of a key enzyme in the formation of the bacterial cell wall.

The effects of reversible inhibitors is shown for both competitive and noncompetitive inhibition compared to the uninhibited enzyme. Note that for competitive inhibition, the velocity V can approach the same Vmax as the unihibited enzyme, but it requires a higher substrate concentration [S]. For the noncompetitive inhibition, a lower Vmax is reached since the inhibiting agent changes the enzyme at a location other than the active site for the substrate.

Using the Lineweaver-Burk plot of the effects of different reversible inhibitors gives plots of the inverses of V and [S]. The noncompetitive inhibitor reduces Vmax without affecting KM, whereas the competitive inhibitor increases KM without affecting Vmax.