Copyright © 2000-2011 Mark Brandt, Ph.D. 1 Introduction to Enzymes Open a can of soda. Breathe out. In both cases, a reaction occurs: the conversion of carbonic acid to carbon dioxide and water. In the case of the soda, the reaction occurs spontaneously and relatively slowly (which is fortunate, because it allows the soda to remain carbonated for quite some time after being opened). On the other hand, if animals had to wait for the reaction to occur, they would have problems associated with build-up of waste carbon dioxide. To avoid this problem, animals have an enzyme, carbonic anhydrase, which accelerates the reaction. (As with most enzymes, carbonic anhydrase catalyzes the reaction in both the forward and reverse directions, and in locations other than the lungs, frequently catalyzes the formation of carbonic acid.) A single molecule of carbonic anhydrase can catalyze the formation of about 106 molecules of CO2 per second. This represents a ~107-fold increase in rate compared to the uncatalyzed reaction. Historical Aspects In the 1880s, Louis Pasteur argued that biological reactions (such as the fermentation of grapes) required living organisms. Eduard Buchner disproved this hypothesis in 1896, when he showed that cell extracts could catalyze the reactions of fermentation. (Buchner won the 1907 Chemistry Nobel Prize). Enzymes were shown to be proteins when James Sumner crystallized the enzyme urease in 1926. More recently, Thomas Czech and others have shown that some RNA molecules exhibit enzymatic activity. General Properties of enzymes Enzymes are catalysts An enzyme enhances the rate of reaction without being consumed in the reaction. The catalytic process requires the enzyme to have a three-dimensional structure; unfolded enzymes do not catalyze reactions. Enzymes have active sites. The active site is the specific part of the enzyme molecule where the reaction occurs. The active site is usually comprised of a relatively small number of residues within the overall enzyme structure. Enzymes do not require harsh conditions to perform elaborate chemistry Enzymes can catalyze reactions under relatively mild conditions. In humans, these conditions are generally 37°C at one atmosphere pressure, with a pH near neutral. This is in marked contrast to organic chemistry reactions, which frequently require fairly extreme conditions. H 2 C O 3 O H 2 C O 2 +Copyright © 2000-2011 Mark Brandt, Ph.D. 2 (Most) enzymes are proteins Although RNA can have catalytic activity, proteins have much more diverse chemistry, because proteins are comprised of 20 amino acids instead of only four nucleotides. In addition, the amino acids have several functional groups that are not present in nucleotides). The chemistry of the amino acid side-chains is critically important when considering enzymatic reactions. Enzymes may have coenzymes or cofactors Some enzymes consist solely of a polypeptide chain. However, amino acid side-chains are not always the best choice for some types of chemistry; enzymes may therefore bind other molecules in order to alter the chemistry of the active site. These other molecules are called “coenzymes” or “cofactors” (the terms are often used essentially interchangeably).1 Metal ions are frequently used as cofactors. For example carbonic anhydrase uses zinc ions as cofactors, and a number of enzymes alter their activity in the presence or absence of metal ions (especially calcium, but also magnesium, zinc, and other ions). Some enzymes require reversibly bound organic compounds for activity. Some enzymes have very tightly bound or irreversibly bound compounds, in which the compound remains associated with the enzyme as long as the enzyme remains in its native conformation). A tightly bound compound that always remains associated with an enzyme and is required for activity is often referred to as a prosthetic group. Prosthetic groups may be covalently or non-covalently associated with the enzyme. The organic or organometallic coenzymes are frequently vitamin derivatives. Some examples of vitamin-derived coenzymes are nicotinamide adenine dinucleotide (NAD), which is derived from niacin (vitamin B3), flavin adenine dinucleotide (FAD), which is derived from riboflavin (vitamin B2), coenzyme A, which is derived from pantothenic acid (vitamin B5), pyridoxal phosphate, which is derived from pyridoxal (vitamin B6), and tetrahydrofolate, which is derived from folic acid. On the other hand, not all coenzymes are derived from vitamins; heme, the prosthetic group of cytochrome P450 enzymes, peroxidases, and hemoglobin is synthesized from amino acids and iron ions. An enzyme lacking its cofactor, coenzyme, or prosthetic group is called an “apoenzyme”; the enzyme with its associated compound is called a “holoenzyme”. Enzymes are specific An enzyme will only catalyze one reaction, or at most, a very small number of closely related reactions. In addition, most enzymes will discriminate between potential substrates. As an example, consider the reaction below: the cytochrome 1Some biochemists define “cofactors” as “metal ions”, and “coenzymes” as “organic” and “organometallic” compounds; others use the terms interchangeably.Copyright © 2000-2011 Mark Brandt, Ph.D. 3 P450 enzyme aromatase catalyzes the conversion of androstenedione to estrone, and of testosterone to estradiol. It will not, however, catalyze a reaction using progesterone. Note that androstenedione, testosterone, and progesterone all have the same structure at the “left” side of the molecule (as shown here using the conventional representation of steroids). The methyl group removed during the reaction (indicated by the arrow) is present in all three of these compounds; the only difference between progesterone and androstenedione is the type of group present at the other end of the molecule. The enzyme aromatase can recognize this difference, and will not use progesterone as a substrate. (Note: the aromatase reaction below is quite complex, and involves other