Enzymes are specialized proteins which are able to conduct chemical reactions under biological conditions. Most enzymes have very specific functions, and convert specific substrates to the corresponding products. Since amino acids are chiral, enzymes also show chirality, and will often act on only one enantiomer of a given substrate. Enzymes are extremely efficient, far more so than chemical catalysts which carry out similar reactions. In addition, enzymes, since they are protein in nature, are dynamic molecules, and must be able to change conformation in order to function properly.
As was mentioned above, enzymes function as biological catalysts. Consider the simple conversion of substrate A to product B, shown below. The substrate molecule A exists with a given energy, and must overcome an energy barrier to be converted to the product B. As A climbs the energy hill, it reaches an energy maximum known as the transition state, A*. The transition state is a reaction intermediate which lies somewhere between A and B; following formation of the transition state, the product B is formed which exists at a new energy level. The difference in energy between A and A* is known as the activation energy, and serves as a barrier to the conversion of A to B.
There are two ways to increase the rate of a chemical reaction. The first is to raise the temperature, but this method can obviously not be used in a biological system. The second way is to use a catalyst. As shown below, enzymes act as biological catalysts by lowering the activation energy. Thus, more molecules can reach the transition state, and thus more product is formed. Enzymes reduce the activation energy by stabilizing the transition state. This occurs because the shape of the enzyme catalytic site is designed to bind to and stabilize the transition state. Thus, the transition state, a fleeting intermediate, binds to the enzyme more easily than substrate or product. Like all catalysts, enzymes are not consumed in the reaction, but are regenerated following each reaction cycle.
Enzyme kinetics and theories of enzyme action were pioneered by Michaelis and Menton, as well as other investigators. According to the Michaelis Menton Hypothesis, enzyme E plus substrate S combine reversibly to form an enzyme-substrate complex ES. Following the formation of ES, the transformation occurs to produce an enzyme-product complex EP. This process may be reversible or irreversible. EP then reversibly dissociates to form product P and the regenerated enzyme E.
There are several possible combinations of enzyme and substrate, giving rise to the comcept of reaction order (see above). We will consider three reaction orders which are important for our purposes. Consider the simple first order conversion of A to P, a unimolecular reaction. As shown above, the disappearance of A depends on the concentration of A, as well as on the first order rate constant k. For a bimolecular reaction such as the conversion of A and B to products P and Q (a second order reaction), the rate of reaction depends on the concentration of A and of B, as well as on the second order rate constant k. In either a unimolecular or bimolecular process, it is possible for the concentration of enzyme to be limiting, a situation known as zero order kinetics. In the case of zero order, the disappearance of A depends only on k, the zero order rate constant. More will be said about these concepts later in this tutorial.
Each enzyme has a specific three dimensional shape which includes an area where the biochemical transformation takes place, called the active site. The active site contains a specialized amino acid sequence that facilitates this transformation. Within the active site are two subsites, the binding site and the catalytic site. The binding site contains residues which cause the substrate to bind to the enzyme by ionic, H-bonding or other electrostatic forces. If a substance fits binds the active site and binds to the enzyme, it is said to have affinity for the active site. The catalytic site contains residues which carry out the actual transformation. There is often considerable overlap between the binding and catalytic sites.
One of the most highly studied enzymes known is the serine protease chymotrypsin. Recall that chymotrypsin cleaves peptides on the carboxyl side of aromatic amino acids (PHE, TRP, TYR). As shown below, there are three residues on the chain which have been identified as participating in the reaction: HIS 57, ASP 102 and SER 195. Notice how the three dimensional structure of the protein, aided by specific disulfide bonds, brings these three residues into proximity.
The chemical mechanism for chymotrypsin-mediated peptide cleavage is shown below. Note that ASP pulls a proton from the neighboring HIS, which in turn pulls the proton from the nearby SER, forming an anion. This form of serine is now more nucleophilic, and attacks the carbonyl of the appropriate amide bond. Mechanisms in which protons are transferred during catalysis are examples of a phenomenon called general acid-base catalysis, and as such, chymotrypsin proceeds via a general base catalysis. Following attack by serine and formation of the transition state, a portion of the peptide containing a new amino terminus is released. A second portion of the peptide remains bound to serine in an ester linkage, which is hydrolyzed to form the second half of the peptide with a new carboxyl terminus. Protons are then transferred to regenerate chymotrypsin in its original form, completing the cycle.
Acetylcholinesterase (ACHE) is a crucial enzyme which deesterifies the ubiquitous neurotransmitter acetylcholine (ACH), thus inactivating it. ACHE is a serine esterase, and has a mechanism similar to chymotrypsin. As shown below, ACHE has an anionic binding site which attracts the positively charged quaternary ammonium group of ACH. A serine then attacks and cleaves the ester. This is another example of general base catalysis, since serine must first be deprotonated by a neighboring HIS. Following ester hydrolysis, the enzyme is quickly regenerated.
There are a number of compounds that are used pharmacologically to inhibit the action of ACHE, thus raising the levels of ACH in the surrounding tissues. Two of the most common of these are the reversible inhibitors neostigmine and physostigmine, shown below. Each drug is positively charged at physiological pH, and is attracted to the anionic site of ACHE. However, instead of forming a serine ester, a carbamoyl group is transferred, forming a serine carbamate [O-C(=O)-N-(R, R')] which is much more slowly hydrolyzed than the ester. While the serine is carbamylated (a few hours), the ACHE is not available to deactivate ACH, and the levels of ACH build up. Interestingly, physostigmine was first isolated from the Calabar bean, which was known as the "ordeal bean" because of its use. Persons alleged to have committed a serious crime were forced to eat a handful of ordeal beans, and presumably if they were guilty, they died! The high amounts of physostigmine in the beans caused a depolarizing block of the diaphragm (from excessive ACH), and the prisoners died from suffocation.
Today, of course, neostigmine and physostigmine are used to treat some forms of glaucoma, because they cause prolonged miosis. This miosis (constriction of the pupils) opens up the canal that drains excess fluid from the eye, resulting in diminished intraocular pressure (IOP). These drugs are also used to treat myasthenia gravis, a disease that results in muscle weakness due to a lack of ACH (the neurotransmitter at the neuromuscular junction). Inhibition of ACHE results in an increase in ACH, and hence increased muscle strength.
There are also a number of irreversible ACHE inhibitors, such as diisopropylfluorophosphate (DFP). These compounds, called organophosphates, form a phosphate ester with ACHE which cannot be hydrolyzed under physiological conditions. Thus, DFP essentially "kills" the enzyme, and activity can only be restored by synthesizing new enzyme. It is possible to administer an antidote for organophosphate poisoning, such as the nucleophilic oxime pralidoxime (2-PAM). 2-PAM has affinity for the ACHE active site, and is sufficiently nucleophilic to attack and cleave the phosphate ester, regenerating ACHE. However, this treatment must be initiated very soon after exposure to the organophosphate.
Enzyme action can be modulated by a number of factors, several of which are discussed below. One of the most fundamental factors affecting enzyme activity is substrate concentration. The effect of substrate concentration on activity is usually expressed using a Michaelis-Menton plot, such as the one shown below, and enzymes which generate such a plot are said to obey Michaelis-Menton kinetics. Michaelis-Menton plots show three distinct regions which correspond to reaction order (discussed above). At low [S], the reaction accelerates as more substrate is added, reflecting first-order kinetics. At high [S], the concentration of enzyme becomes limiting, and additional substrate cannot accelerate the reaction. This situation is known as zero-order kinetics. Finally, there is a transition period between first order and zero order where kinetics are mixed.
If one draws a line across from the level (zero order) region of the plot to the Y-axis, this data point is known as Vmax, the maximum rate of reaction for a given concentration of enzyme. A second kinetic constant is also derived by drawing a line from the Y-axis at 1/2 Vmax to the curve, and then down to the X-axis. This data point, known as the Km, is the concentration of substrate needed to drive the reaction at half Vmax. Each substrate will generate a unique Km and Vmax for a given enzymatic process.
A standard equation used to express the kinetic constants under the Michaelis-Menton hypothesis is aptly called the Michaelis-Menton equation, and is shown below. Later, two other investigators rearranged this equation to generate a second useful equation, the Lineweaver-Burke equation, also shown below.
You should notice two things about the Lineweaver-Burke equation: First, it is in the form y = mx + b, and as such, a plot of this equation will generate a straight line for enzymes obeying simple Michaelis Menton kinetics. In addition, the x and y values for the plot are both inverted, and as such, the plot is often referred to as the double reciprocal plot. The Lineweaver-Burke plot has two advantages over the Michaelis-Menton plot, in that it gives a more accurate estimate of Vmax, and it gives more accurate information about inhibition, as we will see. A typical Lineweaver-Burke plot appears below. Note that Vmax is derived from the y-intercept, and Km can be derived either from the slope, or from extrapolating the line to the negative X-axis.
Enzymes can use Km and Vmax to regulate critical cellular processes, or even to affect tissue distribution of various metabolites. An example of this can be seen in the two similar enzymes hexokinase and glucokinase, which both convert glucose to glucose-6-phosphate, an intermediate in the glucose oxidation pathway which cannot exit the cell. Once glucose enters a cell and is converted to glucose-6-phosphate, it is "trapped" in that cell, and can thus only be used in the metabolism of that particular cell. This is of particular importance in the brain, which cannot synthesize glucose, and thus has an absolute requirement for glucose from the blood.
Although hexokinase and glucokinase conduct the same reaction, they have quite different properties. Hexokinase is found in the brain and in skeletal muscle, and is a regulatory enzyme (i.e. it is inhibited by high concentrations of its product). It also has a higher affinity for glucose, with a Km value of 5 X 10(-5) M. By contrast, glucokinase is found in the liver, and is absent in brain and muscle. It is non-regulatory, and has a lower affinity for glucose (Km = 2 X 10(-2) M). For comparison, note that the normal blood concentration of glucose falls in between these two values, as shown in the graph below.
At normal blood glucose, hexokinase is operating at near Vmax, ensuring that the brain gets an ample supply of glucose. Glucokinase is operating far below its Vmax under these conditions. If blood glucose rises significantly, hexokinase can speed up slightly, but glucokinase speeds up dramatically. In this way, the excess blood glucose is taken up by the liver, and converted to glycogen and fat. If blood glucose falls below normal, hexokinase is still operating near Vmax, while glucokinase is essentially inactive. In this way, a constant supply of glucose is ensured for the brain at all times.
The cell is able to vary the concentration of some enzymes in response to various stimuli. As the concentration of enzyme increases, so does the reaction velocity, as shown in the simple plot below.
There are various methods used by the cell to regulate the concentration of active enzyme. One method is by synthesis and degradation, wherein the cell synthesizes new enzyme when it is needed, and then degrades it when the need is filled. An example of this is the metabolic enzyme ornithine decarboxylase, which has a half-life of 10 minutes in mammalian cells.
The concentration of active enzyme can also be controlled by phosphorylation/dephosphorylation, as shown in the diagram below. A phosphate is added at a apecific amino acid residue (often a tyrosine or a serine), turning the activity of the enzyme on or off. Another enzyme involved in glucose metabolism, glycogen phosphorylase is controlled by phosphorylation state. The B form of the phosphorylase in inactive, and must be phosphorylated by a specific kinase, converting it to the active A form. When the enzyme is no longer needed, it is dephosphorylated back to the B form by a specific phosphatase, rendering it inactive.
Enzyme activity is strongly affected by two additional factors, temperature and pH. Enzymes operate at an optimal temperature, and deviation from this temperature produces a reduction in activity. Most metabolic enzymes function with an optimal temperature near body temperature, but this is not always the case. For example, thermophilic bacteria have metabolic enzymes with optimal temperatures of 85-95 degrees centigrade. Enzymes also operate at a pH optimum, and deviation from this pH produces a reduction in activity. The pH optimum can vary from tissue to tissue (for example, the human enzymes trypsin, pepsin and alkaline phosphatase have pH optima of 8, 1.5-2.5 and 9.5, respectively.
One of the most interesting effects on enzyme action, and of course the most relevant to the practice of Pharmacy, is the presence of inhibitors. An inhibitor is any substance which interferes, either reversibly or irreversibly, with an enzymatic reaction, and of course many drugs are enzyme inhibitors. For our purposes, there are three types of inhibition that are of interest, and each will be covered seperately. The first type of inhibition is known as competitive inhibition. In competitive inhibition, the inhibitor usually resembles the substrate in structure, and competes with it for the active site. As the inhibitor binds to the enzyme, it forms an enzyme-inhibitor complex [EI] which cannot be converted to product. Of course, while this complex is in existence, no substrate can be converted to product, since the active site is occupied. Each competitive inhibitor is associated with a unique kinetic constant, called the Ki, which is simply the dissociation constant for the EI complex, and can be determined by the equation Ki = [E][I]/[EI]. A lower value for Ki denotes a stronger inhibitor. It is important to note that competitive inhibition can be overcome by adding additional substrate.
Consider the Michaelis-Menton plot below, which shows the kinetic effect of adding inhibitor to a reaction at constant [E]. If a concentration X of inhibitor is added, it takes more substrate to reach Vmax, but eventually the inhibitor will be overcome. The same is true at 2X inhibitor, although it takes even longer to reach Vmax. Kinetically, the result is that the Km (the amount of substrate needed to reach Vmax) appears to increase as the [I] increases, giving rise to a value known as Km(app). This is the kinetic hallmark of competitive inhibition: Km increases while Vmax remains unchanged.
The Lineweaver-Burke representation of competitive inhibition gives similar information, as shown below. Note that the slope of the line changes as inhibitor is added, as does the point -1/Km (the X-intercept). However, the Y-intercept (1/Vmax) does not change. The values of Vmax, Km and Km(app) can be readily derived from this plot.
In an instance of competitive inhibition, for a given [I], the Lineweaver Burke Equation becomes:
Note that a factor containing Ki was added to the equation next to the slope, which changes on the inhibition graph, but not next to the Y-intercept, which does not..
The second type on inhibition we will consider is non-competitive inhibition. Most textbooks define non-competitive inhibition as a reversible interaction between inhibitor and enzyme which occurs at a site other than the active site, so that the inhibitor does not compete with substrate for active site binding. For our purposes, we will also include irreversible enzyme inactivators, since they have similar effects, i.e. they decrease the concentration of active enzyme. As seen in the Michaelis-Menton plot below, the Vmax decreases as the [I] increases, but the Km remains unchanged. Because of the nature of the non-competitive interaction, non-competitive inhibition cannot be overcome by adding additional substrate.
The Lineweaver-Burke representation of the same data shows the same effects: -1/Km does not change, while the Y-intercept (and hence Vmax) does. The values of Vmax and Km can be readily derived from this plot.
Since both the slope and the Y-intercept change as inhibitor is added, the Lineweaver-Burke Equation for non-competitive inhibition becomes:
The last type of inhibition we will consider is called
Because the lines are parallel, only the Y-intercept changes, and the Lineweaver-Burke Equation for uncompetitive inhibition becomes:
Some enzyme inhibitors act as irreversible inactivators of the target enzyme, forming a covalent intermediate in the active site which cannot be readily removed. In some cases, the inhibitor enters the active site as an inert species, and is converted by the enzyme to a latent reactive intermediate that alkylates the catalytic site, inactivating the enzyme. These inhibitors are known as mechanism-based inhibitors (sometimes also referred to as suicide substrates or Kcat inhibitors, since they depend on catalysis for their effect). A true mechanism-based inhibitor must satisfy the following criterion:
Kinetic constants are determined for mechanism-based inactivators in the following way: The time dependent decay of enzyme activity is monitored at several concentrations of the inhibitor, and the results are plotted on a semilog scale.
The slope for each line is determined, and represents the rate constant k(obs), and these values are replotted (1/k(obs) VS 1/[I]) using a Kitz-Wilson plot. The y-intercept data point is 1/k(inact), where k(inact) represents the rate constant for inactivation. The x-intercept data point is -1/Ki, where Ki is a value similar to Km, and represents the affinity of the inhibitor for the active site.
Ideally, each molecule of the inactivator should destroy one molecule of enzyme, but in practice, this is not the case. Some of the laten reactive species formed in the active site forms a false product (e.g. by reaction with a water molecule), and this transformation does not result in inactivation. The ratio between false catalysis k(cat) and inactivation k(inact) is called the partition ratio, and can be determined experimentally.
Another way to control enzyme activity is a method we have discussed before, called allosteric control. The simplest example of this is a concept known as feedback inhibition. Let us assume that a substrate A is converted to a product B, which is then converted by additional enzymes to C, D, E, etc. Eventually, a final product P is synthesized. Let us further assume that the enzyme converting A to B is a committed step (i.e. the product can only be used to make P) and rate limiting (i.e. it is the slowest step in the pathway). If this pathway exhibits feedback inhibition, P (or some other intermediate near the end of the pathway) will downregulate the enzyme converting A to B, thus shutting down the pathway before the committed intermediate B is formed. This is the phenomenon known as feedback inhibition. An example of this appears below. The enzyme aspartate transcarbamylase makes a committed intermediate, carbamyl aspartate, which is then used to make cytosine triphosphate (CTP), an important nucleotide used in DNA and RNA synthesis. CTP can feedback and shut off aspartate transcarbamylase, effectively shutting down the CTP pathway.
Allosterism simply produces a conformational change in the enzyme that changes its activity. The enzyme can be more active (positive allosterism) or less active (negative allosterism). It may have one effector (monovalent) or more than one (polyvalent), and the effector may be the substrate (homotropic) or some other molecule (heterotropic). We have previously discussed cooperativity, which is a special case of allosterism that pertains to enzymes with multiple active sites. It is important to point out that allosteric enzymes do not obey Michaelis-Menton kinetics, since they produce sigmoid curves that include a lag time for enzyme activation, as shown below.
This seems like a good place to introduce the comcept of isozymes, which are multiple forms of an enzyme that conduct the same catalytic reaction. These isozymes usually are coded for by different genes, and have slightly different amino acid sequences or subunit makeup. For this reason, they can usually be seperated by electrophoreisis. An excellent example of an isozyme is lactate dehydrogenase (LDH), a tetrameric enzyme which converts pyruvate to lactate under aerobic conditions. LDH can be comprised of two types of subunit, the M subunit, which has a low Km for pyruvate and a high rate of conversion to lactate, and the H subunit, which has a high Km for pyruvate and a low rate of conversion to lactate. In skeletal muscle, LDH is mostly M4 or M3H, and this allows the muscle to rapidly convert pyruvate to lactate, producing energy when oxygen supplies are low. Heart muscle contains mostly H4 and H3M, and as such does not convert pyruvate to lactate under anaerobic conditions. This is one reason why heart muscle is dependent on oxygen for proper function.
Some enzymes are synthesized in an inactive form called a zymogen or proenzyme form. These enzymes must be processed (cleaved) by a protease before they become active. The peptidases
We have alluded to the fact that enzyme concentration can be controlled in vivo by synthesis and degradation. The mechanism underlying this form of control has been elucidated, and is known as the Jacob-Monod Theory. It was first discovered in the bacteria Escherichia coli. Consider the DNA structure below; this structure contains an operon, which is a segment of DNA that codes for a group of related enzymes or other proteins. To be specific, E. coli DNA contains a region called the lac operon, which codes for the synthesis of three enzymes, beta-galactosidase, permease and a specific acetylase. Beta galactosidase is an enzyme which converts lactose (a sugar comprised of glucose and galactose) to glucose, which is the preferred energy source for E. coli.
Near the lac operon are three other short regions of DNA: a regulator gene, which codes for the synthesis of a repressor protein, a promoter site which signals beginning of a region that needs to be read and used to make protein, and an operator site which is used to bind the repressor protein, as we shall see.
Under normal conditions, the repressor protein is made from the regulator gene; if no lactose is present, the repressor protein binds to the operator site. The enzymatic machinery that is used to make messenger RNA (m-RNA) from DNA, the DNA dependent RNA polymerase binds at the promoter site and attempts to make mRNA. However, it cannot move past the repressor, so mRNA synthesis is shut down on this gene. When lactose is present, it binds to the allosteric site of the repressor protein, and the protein changes conformation and falls off the operator site. DNA dependent RNA polymerase is now free to move along the DNA strand, and the DNA is used to make mRNA in a process called transcription. This mRNA is later used to make protein in a process called translation. Thus, when E. coli encounters lactose, the lac operon is "turned on", and beta-galactosidase is synthesized.
You should be aware that there is a regulatory body known as the Enzyme Commission which is responsible for the classification of all enzymes. Each enzyme is assigned a specific code number which can be used to describe its function. For example, the Enzyme Commission number for creatine kinase is shown below:
Oxidoreductases are enzymes which catalyze oxidation or reduction reactions. An example is the reductase (dehydrogenase) alcohol dehydrogenase, a human enzyme which converts ethanol to acetaldehyde. A second enzyme known as aldehyde dehydrogenase then converts acetaldehyde to acetyl CoA. Oxidoreductases often require a cofactor, in this case NAD+, which accepts the hydrogens released during oxidation.
Oxidases are so named only if oxygen is used as an acceptor molecule. An example is glucose oxidase, which converts sugars such as glucose to the corresponding gluconic acid.
Transferases are enzymes which transfer a functional group from a donor molecule to an acceptor molecule. A common transferase is a methyltransferase, which transfers a methyl group from S-adenosylmethionine to some acceptor. The example shown below is catechol-O-methyltransferase, which is an enzyme involved in the catabolism of the neurotransmittwers epinephrine and norepinephrine.
One very important example of a transferase enzyme is a transaminase. A transaminase takes one amino acid and one alpha keto acid, and converts it to a second amino acid and a corresponding alpha keto acid, thus apparently transferring an amino functional group from one molecule to another. This allows for the interconversion of certain amino acids, and also allows amino acids to enter into glucose metabolism, as we shall see later.
A transferase you will see again and again in biochemistry is the kinase, which acts to transfer a phosphate from the high energy phosphate ATP to the substrate. There are numerous kinases that play a variety of critical roles in cellular metabolism.
Hydrolase enzymes catalyze biological hydrolysis reactions, i.e. they break bonds while adding the elements of water across the bond being broken. Lipases, phosphatases, acetylcholinesterase and proteases are all examples of hydrolases.
Lyases catalyze the cleavage of C-C, C-O and C-N bonds by a means other than hydrolysis. An example is the important enzyme DOPA decarboxylase, which is a critical step in the synthesis of the biogenic amines epinephrine and norepinephrine.
Isomerases are simply enzymes which catalyze intramolecular rearrangements. Epimerases and racemases are examples of this class.
Ligases catalyze the formation of C-O, C-S, C-N or C-C bonds coupled with the hydrolysis of the pyrophosphate bond of ATP. The phosphate may or may not be covalently bound to the product.
Cleland has devised a standardized way of referring to bisubstrate (Bi-Bi) enzymatic reactions, which make up 60% of all enzymatic transformations. The substrates, products and stable enzyme forms are denoted as follows:
The first important type of bi-bi reaction is known as sequential, which means that all substrates must add to the enzyme before any reaction takes place. The sequential bi-bi can be ordered, meaning that the substrates add in a specific order, or random, meaning that both substrates must add, but in no particular order. A Cleland diagram for an ordered sequential bi-bi is shown below.
The second important type of bi-bi is the so-called ping-pong bi-bi, as shown below. Note that one substrate adds to the enzyme, producing the E-A complex, but then the enzyme is covalently altered to form a complex between a stable enzyme form F and the first product P. P is then ejected, and a second substrate B adds. The group originally transferred to E to form F is then transferred to B, forming Q, and regenerating the enzyme form E. Finally, Q is ejected and the cycle is complete.
The enzyme acid phosphatase is released due to cell destruction in patients suffering from prostate carcinoma. The laboratory assay for acid phosphatase is shown below. p-Nitrophenol phosphate is converted in vitro by acid phosphatase to p-nitrophenol, which has a yellow color. Thus, this is a colorimetric assay used to support the diagnosis of prostate cancer. A similar enzyme, alkaline phosphatase, can also be assayed in this manner, except that the reaction is carried out at pH 9-10. Elevated alkaline phosphatase is observed in obstructive jaundice, cirrhosis, osteogenic sarcoma, rickets, Paget's disease and other bone disorders.
The enzyme alpha amylase, which is a digestive enzyme produced by the pancreas, is elevated in the serum in pancreatitis or pancreatic cancer. It is also assayed by a colorimetric procedure.
Creatine phosphokinase (CPK) is an enzyme found mostly in skeletal and cardiac muscle. This enzyme is elevated after a myocardial infarction, and can be measured either by a complex assay, or by electrophoreisis.
Lactate dehydrogenase (LDH) is elevated in a number of disease states, including myocardial infarction. The assay involves the in vitro conversion of pyruvate to lactate, and requires the cofactor NADH. NADH, which has a UV absorbance at 340 nm, is converted to NAD+, which does not. Therefore, assay of this enzyme involves observing the decrease in absorbance at 340 nm.
There are two important transaminases that are used in the diagnosis of disease, serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT). SGOT is elevated in myocardial infarction, and also when there is liver damage or disease present. SGPT is elevated only in liver disease.
Finally, the enzyme alpha-hydroxybutyrate dehydrogenase is elevated during myocardial infarction, and is measured by monitoring the decrease in absorbance at 340nm, as described above.
The graph below shows a typical enzyme profile from a patient who has recently had a myocardial infarction. Notice that the various enzymes are elevated at different time intervals, a phenomenon which aids in the diagnosis of this disease.
Enzyme defects are often the result of genetic idiosyncracies in certain patients. An important example is the condition known as phenylketonuria, the biochemistry of which is shown below. Normally, a great deal of phenylalanine is converted in vivo to tyrosine, which is then used for protein synthesis or in the synthesis of neurotransmitters. However, in patients with phenylketonuria, the enzyme that forms tyrosine, phenylalanine-4-monooxidase is lacking. In this case, a minor pathway takes over, producing large amounts of phenylpyravate. When phenylpyruvate is formed in excess, it enters the CNS, where it can cause mental retardation.
The treatment for this syndrome is a low phenylalanine diet, and infants with this condition must be placed on a phenylalanine-free formula (Lofenelac®). Adults with this syndrome must avoid aspartame (NutraSweet®), since it contains phenylalanine.
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The reaction must be time-dependent and pseudo-first order in this regard.
The reaction must be irreversible, such that the adduct cannot be removed by dialysis.
There must be 1:1 stoichiometry in the inactivation step.
The enzyme should be protected from inactivation by previously added substrate, or by adding a reversible inhibitor, prior to adding the inactivator.
Classification of Enzymes
Oxidoreductases
Transferases
HydrolasesLyases (Desmolases)
IsomerasesLigasesCleland Nomenclature for Enzymes
Substrates are lettered A, B, C and D, in the order that they are added to the enzyme.
Products are lettered P, Q, R and S, in the order that they leave the surface of the enzyme.
Stable enzyme forms are lettered E, F and G, in the order that they occur.
The number of reactants in the reaction are designated by the terms Uni, Bi, Ter and Quad.
Enzymes in the Diagnosis of Disease
Enzyme Defects
