Enzymes Flashcards

Question

HOW DO ENZYMES WORK?

Answer 1

Biological catalysts increase the rate of a chemical reaction, permitting reactions to occur that would otherwise be so slow as to be incompatible with life. Mammalian enzymes have evolved to catalyze reactions under physiological conditions, that is, at 37°C and usually at a pH near neutrality. They commonly accelerate reactions by factors of lo6 to 10" and are usually highly specific for their substrates. The active site of an enzyme is the pocket in the protein where the substrate or substrates are bound. Substrates are bound to enzymes in what is referred to an enzyme-substrate (ES) complex by multiple weak (usually noncovalent) interactions, particularly ionic and hydrogen bonds. Binding of substrates to the enzyme's active site stabilizes the reaction intermediate or transition state, thereby decreasing the amount of activation energy required for the reaction to occur (Fig. 2- 1). Theoretically, all chemical reactions are reversible to some extent. Enzymes catalyze both the forward and reverse reactions. ## Footnote What Determines the Direction in Which Reversible Reactions Proceed? Irreversible Reactions Isozymes Are Different Proteins That Catalyze the Same Reaction

Answer 2

An example of a reversible reaction is the one catalyzed by lactate dehydrogenase: lactate + NAD' + pyruvate + NADH + H' Whether one starts with the substrates (shown on the left) or the products (on the right), the lactate dehydrogenase reaction, like all reactions, will eventually reach an equilibrium or steady-state condition. At equilibrium, the relative proportion of reactants on the left and products on the right will be determined by the change in free energy of the reaction (AGO'); in other words, the reaction will proceed in the direction that releases energy (AGO' < 0) rather than one that requires a net input of energy. For reversible reactions, the major factor that determines the rates of reactions in the forward and reverse directions is the relative concentration of substrates and products. For reactions like the one catalyzed by lactate dehydrogenase, the direction of the reaction is determined primarily by the NADH/NAD+ ratio. Thus, when exercising muscle produces more NADH than can be utilized by the mitochondria1 oxidative phosphorylation system, the buildup of NADH drives lactate dehydrogenase to produce lactate from pyruvate. Conversely, when hepatocytes are actively utilizing NADH for ATP production via oxidative phosphorylation, the NADH level falls and the concentration of NAD+ increases, thereby causing lactate dehydrogenase to generate pyruvate from lactate.

Answer 3

There are many reactions that are essentially irreversible under physiological conditions. These irreversible reactions are exergonic, meaning that they give off significant energy. Biochemists consider a reaction to be irreversible when the free- energy change (AGO’) is -4 kcal/mol or more negative. An example of a physiologically irreversible reaction is that catalyzed by glucose 6-phosphatase: glucose 6-phosphate + H2O -> glucose + Pi The reverse reaction, that is, the formation of glucose 6-phosphate, would require the input of significant energy. Neither glucokinase nor hexokinase, the two enzymes that catalyze the synthesis of glucose 6-phosphate from free glucose, can directly reverse the reaction catalyzed by glucose 6-phosphatase. Instead, both of these enzymes utilize the energy associated with one of the high-energy bonds of ATP to phosphorylate glucose: glucose + ATP -+ glucose 6-phosphate + ADP Acetyl-CoA carboxylase is another enzyme that utilizes the high-energy y -phosphate bond of ATP to drive a reaction that would be irreversible without the participation of ATP: acetyl-CoA + CO2 + ATP + H20 -> malonyl-CoA + ADP + Pi In this case, the terminal (y) phosphate of ATP (Fig. 1-2) is not incorporated into the product of the reaction. Instead, two reactions (hydrolysis of ATP and carboxyl- ation of acetyl-CoA) are coupled, with the favorable (energy-yielding or extqonic) hydrolysis of ATP being used to drive the unfavorable (energy-requiring or ender- gonic) carboxylation of acetyl-CoA.

Answer 4

As described above, glucokinase and hexokinase both catalyze the synthesis of glucose 6-phosphate from glucose and ATP. However, the two enzymes differ with regard to both their catalytic properties and their protein structures, and are therefore called isozymes or isoenzymes. Hexokinase, the isozyme present in almost every cell of the body, has a high affinity for glucose and is therefore active even at relatively low concentrations of glucose (Fig. 2- 12). By contrast, glucokinase, which is found primarily in liver, is relatively inactive at low concentrations of glucose. Glucokinase has a higher maximal activity than hexokinase and is able to respond to increased blood glucose concentrations by rapidly synthesizing glucose 6-phosphate. Biochemists quantify these differences by indicating that glucokinase has both a higher Vmax (maximal reaction velocity) and a higher Km (the substrate concentration required to support half-maximal activity) than hexokinase. As shown in Figure 2- 12, the K, of hexokinase for glucose is 0.01 mM, while the lower affinity of glucokinase for glucose is reflected by its higher K, of 5 to 10 mM. This difference in K,, values between the two isozymes permits the liver to remove glucose rapidly from the blood when the glucose concentration is high, while leaving glucose available for glucose- dependent tissues (e.g., red blood cells, brain) when the body’s glucose reserves are low.

Answer 5

Regulation of the functioning of the many enzymes in the body is central to co- ordinating the multiple pathways of metabolism and maintaining homeostasis. One key mechanism for regulating the level of activity of a particular enzyme in a cell is regulation of gene expression, since if an enzyme is not synthesized in the appropriate cell or at a particular time, the reaction it catalyzes will not occur. The activities of existing enzymes are themselves also regulated, both by intracellular availability of metabolites and by covalent modifications (e.g., phosphorylation). In addition, many pharmaceutical agents act by inhibiting the activity of one or more enzymes. For example, patients who have elevated plasma cholesterol levels may be prescribed one of the statins, a class of drugs that inhibit HMG-CoA reductase, which catalyzes the rate-limiting step in cholesterol synthesis. The major mechanisms for regulation of enzymatic activity are described below. ## Footnote Competitive Enzyme Inhibition Noncompetitive Enzyme Inhibition Allosteric Regulation Regulation of Enzyme Activity by Covalent Modification Induction of Enzyme Synthesis

Answer 6

Competitive inhibition occurs when a molecule that is not a substrate for the enzyme in question, but which is structurally similar to the substrate, competes with the substrate and blocks its binding to the active site of the enzyme. Occupation of the active site by the inhibitor decreases the activity of the enzyme, particularly when the concentration of the substrate is low relative to that of the inhibitor. Many pharmaceutical agents are competitive inhibitors of specific enzymes. For example, dicumarol inhibits catalysis involving vitamin K. Since many enzymes in the blood-clotting cascade are activated by y -carboxylation, dicumarol acts as an anticoagulant that reduces the risk of thrombus formation. In some cases, two different molecules may both be substrates for the same enzyme, with each acting as a competitive inhibitor of the metabolism of the other. One such example is alcohol dehydrogenase, which catalyzes the oxidation of both ethanol and methanol: ethanol + NAD+ -> acetaldehyde + NADH + H+ methanol + NAD+ -> formaldehyde + NADH + H+ Although methanol itself is intoxicating, it is the metabolites of methanol (formalde- hyde and formic acid) that are responsible for the blindness and death that result from methanol poisoning. One treatment for acute methanol poisoning involves in- travenous administration of ethanol (plus glucose). Ethanol acts as a competitive inhibitor of the conversion of methanol to formaldehyde, thereby preventing ac- cumulation of toxic metabolites until the methanol can be cleared by the kidneys. Glucose is administered to correct the hypoglycemia caused by ethanol.

Answer 7

A noncompetitive inhibitor binds to its target enzyme and cannot be displaced by excess substrate. Thus, this type of inhibitor diminishes the fraction of the enzyme pool that is catalytically competent. Aspirin's action as a noncompetitive inhibitor is the major reason that it is a drug of choice for long-term therapy to decrease the risk of cardiovascular crises. Aspirin is a member of a class of drugs called nonsteroidal anti-inflammatory agents (NSAIDs) that inhibit the cyclooxygenase isozymes, thereby decreasing thromboxane production and platelet aggregation. Aspirin is an irreversible inhibitor since the molecule covalently acetylates a serine residue at the active site of the enzyme, inactivating cyclooxygenase permanently. By contrast, the inhibitory actions of other NSAIDs, such as ibuprofen, are attributable to reversible, noncovalent interactions between drug and enzyme. The reaction of aspirin with cyclooxygenase is particularly effective in platelets because platelets are incapable of synthesizing new enzyme protein. Some of the deleterious effects of heavy metals, such as mercury and lead, re- sult from their actions as noncompetitive enzyme inhibitors. For example, mercury inhibits glyceraldehyde 3-phosphate dehydrogenase, an enzyme in the glycolytic pathway, while lead inhibits heme synthesis.

Answer 8

Allosteric regulation, as this phenomenon is called, provides a mechanism by which enzymatic activities can be modulated by compounds that have little or no structural similarity to the substrate(s) but which instead, reflect the overall metabolic state or needs of the cell. Allosteric enzymes usually exhibit sigmoidal (S-shaped) kinetic curves rather than simple hyperbolic curves. Activators of allosteric enzymes shift the V vs. S curve to the left, whereas allosteric inhibitors shift the curve to the right. ## Footnote End-Product Inhibition. Regulation by Molecules That Signal the Availability of Precursors. Regulation by the Energy Charge of the Cell

Answer 9

**End-Product Inhibition**. There are many instances in which the final endproduct of a multienzyme metabolic pathway is an allosteric inhibitor of an enzyme that catalyzes an early and irreversible step of the pathway. This form of allosteric regulation prevents accumulation of additional end product and of metabolic intermediates once a cell has sufficient supplies of that metabolic end product. Examples of this are seen in the pathways that generate heme, long-chain fatty acids, and cholesterol, where the end products inhibit delta-aminolevulinic acid synthase, acetyl-CoA carboxylase, and HMG-CoA reductase, respectively. **Regulation by Molecules That Signal the Availability of Precursors.** Allosteric regulation provides a mechanism by which flux through a particular pathway can be rendered responsive to the overall nutritional state and needs of the cell. One such important small regulatory metabolite is citrate, an intermediate in the tricarboxylic acid cycle. Citrate allosterically stimulates liver cells to synthesize both fatty acids and glucose (gluconeogenesis) while inhibiting the breakdown of glucose by glycolysis. **Regulation by the Energy Charge of the Cell.** Allosteric mechanisms also serve to regulate many metabolic pathways in response to a high ATP/ADP ratio, which is indicative of a plentiful supply of energy, or conversely, to high concentrations of ADP and adenosine 5’-monophosphate (AMP), which occur when ATP supplies have been depleted. An enzyme whose activity is regulated by the energy charge of the cell is the muscle isozyme of glycogen phosphorylase, which releases glucose (as glucose 1-phosphate) from glycogen stores. AMP is an allosteric activator of glycogen phosphorylase, and ATP is an allosteric inhibitor of the enzyme.

Answer 10

1. Phosphorylation/Dephosphorylation. 2. Hydrolytic Cleavage of Inhibitory Peptides 3.

Answer 11

The most common covalent modification utilized in regulating human metabolism is the reversible phosphoryl- ation of enzyme proteins. In most cases, phosphorylation is the result of a hormone- stimulated signal-transduction cascade, thus providing a mechanism by which intracellular enzymatic activity can be modulated in response to intercellular signaling. For example, glucagon and epinephrine both stimulate the activity of a serine-threonine protein kinase called protein kinase A (PKA). Briefly, this particu- lar signaling pathway involves binding of glucagon or epinephrine to its respective transmembrane receptors, activation of a GTP-binding or G-protein, and activation of adenylyl cyclase, an enzyme that synthesizes cyclic AMP (CAMP) from ATP ATP -> CAMP + PPi CAMP is an allosteric activator of PKA. Once activated, PKA uses ATP to phos- phorylate specific serine and threonine residues on critical enzymes that regulate the flux of intermediates through key metabolic pathways. As discussed in more detail in Chapter 8, PKA-catalyzed phosphorylation activates enzymes involved with glycogen breakdown (glycogen phosphorylase). PKA-catalyzed phosphorylation also activates enzymes involved with mobilization of triacylglycerol stores and gluconeogenesis. Concurrently and conversely, PKA-catalyzed protein phosphorylation inhibits enzymes involved in glycogen and fatty acid metabolism. The simultaneous phosphorylation of multiple enzymes provides a coordinated response to the body’s need to mobilize endogenous fuels during fasting or in response to stress. Protein phosphorylation catalyzed by PKA can be reversed. Among the many effects of stimulation of cells by insulin is the activation of a signal-transduction cascade that results ultimately in the activation of protein phosphatase- 1. Protein phosphatase-1 hydrolyzes the phosphate moieties from phosphoserine and phospho- threonine residues of many enzymes, thereby reversing the activation or inactivation that occurred when those enzymes were phosphorylated. Accordingly, insulin reverses the metabolic effects of glucagon and epinephrine, and switches the direction of key metabolic processes to meet the body's needs in the fed state when there is active synthesis of triacylglycerol in adipocytes and hepatocytes as well as synthesis and storage of glycogen in muscle and liver.

Answer 12

A number of enzymes, particularly digestive enzymes synthesized in the pancreas and the liver-synthesized proteases of the blood-clotting cascade, are secreted from their sites of synthesis in an inactive or zymogen form. Activation requires proteolytic hydrolysis of the pro- enzyme and release of a polypeptide, which then permits the remaining polypeptide fragment to alter its three-dimensional structure to one in which its active site and associated substrate binding pocket are configured correctly for catalysis. Thus, the digestive enzyme trypsin is secreted from the pancreas in the form of an inactive precursor, trypsinogen. Once in the lumen of the small intestine, a brush-border protease called enteropeptidase hydrolyzes one peptide bond within the trypsinogen molecule, thereby releasing the inhibitory peptide and generating active trypsin. Secretion of trypsin in its zymogen form limits its activity to the digestive tract, thus protecting the pancreas and pancreatic duct from proteolytic damage.

Answer 13

Hormonal regulation of enzymatic activity can also occur through stimulation or inhibition of transcription of genes that encode key metabolic enzymes. Hydrocortisone, a glucocorticoid hormone synthesized by the adrenal cortex, acts by entering the cell and binding to certain proteins in the cytosol that serve as glucocorticoid receptors. The hydrocortisone-glucocorticoid receptor complex then translocates to the nucleus, where it binds to specific hormone-response elements in DNA. The actions of hydrocortisone include induction of the synthesis of enzymes involved with gluconeogenesis, mobilization of adipose triacylglycerol, and degradation of muscle proteins. Hydrocortisone thus plays a major role in mediating long-term adaptations in the activities of metabolic pathways in response to starvation, sepsis, and stress.

Answer 14

1. Vitamin Deficiencies a. Scurvy b. Pellagra 2. Inborn Errors of Metabolism 3. Vitamin-Dependency Diseases 4. alpha1 -Antitrypsin Deficiency 5. Pancreatitis 6. Enzymes as Markers of Disease

Answer 15

Since vitamins are crucial components of many enzyme cofactors, an inadequate concentration of one or more of these essential dietary substances can result in **impaired enzymatic activity**. Vitamin deficiencies result from inadequate dietary intake or from impaired absorption or recycling of a vitamin. Impaired absorption of vitamins K and B12. Unlike many of the other vitamin-based cofactors that attach to their respective enzymes through noncovalent bonds, Biotin is covalently attached to lysyl residues of the enzymes with which it functions. Biotin deficiency can result from inadequate activity of the enzyme biotinidase, which normally hydrolyzes the biotinyl-lysyl bond and releases free biotin, thus permitting recycling of biotin when biotin-containing enzymes are degraded. A deficiency of biotin can also be induced by consumption of raw eggs, which contain avidin, a protein that binds biotin very tightly, thereby preventing absorption of biotin from the gut. Biotin deficiency reduces the activities of all four biotin-dependent enzymes: pyruvate carboxylase, acetyl-CoA carboxylase, propionyl-CoA carboxylase, and P-methylcrotonyl-CoA carboxylase. Dietary deficiencies of particular vitamins usually result from restricted diets. In each case, there is impaired activity of all of the enzymes that utilize the particular vitamin-derived cofactor, and ultimately development of a specific vitamin-deficiency disease. Thus, deficiency of folic acid results in megaloblastic anemia and is associated with congenital neural tube defects, whereas the peripheral neuropathy and cardiac manifestations of beriberi are caused by a dietary deficiency of thiamine.

Answer 16

Scurvy is the result of a dietary **deficiency of vitamin C **(ascorbic acid), which is usually obtained from fresh fruits and vegetables, especially citrus fruits (e.g., oranges, grapefruit, limes), cabbage, mangoes, and tomatoes. Ascorbic acid is a cofactor in various hydroxylation reactions including hydroxylation of specific proline and lysine residues of procollagen and the hydroxylation of dopamine to form norepinephrine. Scurvy is primarily a disease of defective collagen synthesis, and is characterized by bleeding gums, hemorrhages, and impaired wound heeling. Ascorbic acid also plays an important non-cofactor role as an antioxidant; it regenerates the reduced forms of other antioxidants, such as vitamin E and glutathione, as well as inactivating potentially harmful reactive oxygen species and nitrogen radicals. Scurvy has been recognized since ancient times, and many indigenous cultures are known to have had remedies utilizing local plant sources, including teas brewed from pine needles. Scurvy was particularly rampant among European sailors on long ocean voyages.

Answer 17

A **deficiency of niacin** results in pellagra, which is characterized by dermatitis (especially in areas of the skin exposed to sunlight), diarrhea, dementia, and ultimately-if untreated-death. Since niacin is the vitamin component of NAD+ and NADP+, which are cofactors in numerous oxidation-reduction reac- tions, it is involved with essentially all of the major metabolic pathways including glycolysis, P-oxidation of fatty acids, the TCA cycle, electron transport and oxidative phosphorylation, and the synthesis of fatty acids and cholesterol. Pellagra was endemic in the American Southeast between the two world wars, primarily among poor mill workers, who consumed a limited diet consisting primarily of corn (maize) and lard. The niacin in maize has relatively low bioavdability unless the maize is treated with alkali. Europeans adopted corn as a crop but not the native tradition of grinding corn with lime (calcium oxide or calcium carbonate). Niacin is unique among the B vitamins in that the dietary requirement for this vitamin can be partially satisfied by endogenous synthesis of niacin from the essential amino acid tryptophan; unfortunately, maize also happens to be a relatively poor source of tryptophan. Prevention of pellagra was accomplished through public health measures, particularly the fortification of cereal products (e.g., bread, biscuits, pasta) with niacin. Ironically, these measures were delayed for many years because public acceptance of pellagra as a disease of malnutrition was hampered by the eugenics movement, which stereotyped the victims of pellagra in the U.S. South as inherently inferior human beings.

Answer 18

Inborn errors of metabolism are **genetic disorders** resulting from **partial loss of function or from null mutations (complete absence of activity) of genes coding for particular enzymes**. Examples of inborn errors of metabolism include phenylketonuria (PKU, caused by a lack of phenylalanine hydroxylase), medium-chain acyl-CoA hydrogenase deficiency, and glucose 6-phosphate dehydrogenase deficiency. Other examples of inborn errors are the lysosomal storage diseases, which result from the loss of function of acid hydrolases required for lysosomal digestion of glycosaminoglycans, glycolipids, sphingomyelin, and glycogen.

Answer 19

Inborn errors of metabolism often result from the synthesis of mutated enzymes, which have a decreased affinity (increased Km) for their coenzyme or prosthetic group. In such cases the patient can often be treated successfully with exceptionally high intakes-or megadoses-of the vitamin precursor. For example, **cystathionine synthase** is a pyridoxal phosphate-dependent enzyme that synthesizes cystathionine from homocysteine and serine. Some forms of **cystathionine beta-synthase deficiency are responsive to treatment with pyridoxine (vitamin B6)**. Similarly, some persons who are deficient in **pyruvate dehydrogenase improve with thiamine therapy**.

Answer 20

alpha1-Antitrypsin is a plasma glycoprotein that inhibits the activity of **elastase** and has inhibitory activity against a number of other serine proteases. Synthesized in and secreted by the liver, alpha1-antitrypsin’s major physiological function is to inhibit elastase released by neutrophils in the lung. In the absence of this bloodborne **protease inhibitor**, there is destruction of elastin in pulmonary alveoli, resulting in chronic obstructive pulmonary disease or emphysema. The disease occurs much earlier and is more severe in people who smoke, and some people with alpha1-antitrypsin deficiency also develop cirrhosis of the liver.

Answer 21

Pancreatitis is an inflammation of the pancreas which can result from a number of conditions, including gallstones, chronic alcoholism, and the blockage of the pancreatic duct that can occur in cystic fibrosis. Damage to pancreatic cells results in premature activation of **digestive proteases** within the pancreas and resulting auto-digestion of the pancreas. Elevated serum levels of pancreatic enzymes, particularly **pancreatic lipase and amylase**, are a laboratory-based diagnostic criterion of acute pancreatitis.

Answer 22

Many tissues produce enzymes that are **relatively cell-specific**. Because these enzymes are released into the circulation as a result of tissue damage, assays of the levels of certain enzymes in blood can provide useful diagnostic information. Probably the most widely requested plasma enzyme assays are those for alanine aminotransferase (ALT) and aspartate aminotransferase (AST), both of which are present in high concentration in hepatocytes. When these cells are injured, for example by viral hepatitis or acetaminophen overdose, ALT and AST are released into the blood. As indicated above, elevated serum levels of pancreatic lipase and amylase are indicative of acute pancreatic disease. The level of the MB isozyme of creatine kinase (CPK), a marker for myocardial infarction, often rises rapidly in the plasma of a person who has experienced a heart attack. An increased level of the MM- isozyme of CPK is usually indicative of injury to skeletal muscle. Other examples of tissue-specific enzyme markers of cellular injury include bone-specific alkaline phosphatase (b-ALKP), which serves as a marker of bone turnover in patients with osteoporosis or Paget’s disease, and acid phosphatase, which is elevated in plasma of patients with metastatic cancer of the prostate.