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The easy way to take the confusion out of organic chemistry Organic chemistry has a long-standing reputation as a difficult course. Organic Chemistry I For Dummies takes a simple approach to the topic, allowing you to grasp concepts at your own pace. In general, the formation of these hydrogen bonds leads to the secondary structure of a protein. The secondary structure is the result of many hydrogen bonds, not just one. The hydrogen bonds are intramolecular, that is between segments of the same molecule, as shown in Figure 5—3: Figure Hydrogen bonding between two peptide bonds.
Secondary structures may be only a small portion of the structure of a protein or can make up 75 percent or more. Each turn consists of 3. These turns allow hydrogen bonding between residues spaced four apart. Every peptide bond participates in two hydrogen bonds: one from an NH to a neighboring carbonyl, and one from a neighboring NH to the carbonyl Figure Structurally, the helices may be either right-handed or left-handed see Chapter 3 for more on handedness.
Essentially all known polypeptides are right-handed. Slightly more steric hindrance is present in a left-handed helix, and the additional steric hindrance makes the structure less stable. A group of isoleucine residues disrupts the secondary structure because of the steric hindrance caused by their bulky R groups.
The small R group of glycine, only an H, allows too much freedom of movement, which leads to a destabilization of the helix. Other residues that destabilize the helix, for similar reasons, are lysine, arginine, serine, and threonine. Here, the primary structure is extended instead of tightly winding into a helix. Again, hydrogen bonds are the source of these structures. The strands are different parts of the same primary structure. In the parallel structure, the adjacent polypeptide strands align along the same direction from N-terminal end to C-terminal end.
In the anti-parallel structure, the alignment is such that one strand goes from N-terminal end to C-terminal end, while the adjacent strand goes from C-terminal end to N-terminal end Figure The hydrogen bonding pattern in the parallel structure is the more complicated.
Here, the NH group of one residue links to a CO on the adjacent strand, whereas the CO of the first residue links to the NH on the adjacent strand that is two residues down the strand. If the arrows point in the same direction, it is the parallel structure, and if they point in opposite directions, it is the anti-parallel structure.
The sheets are typically 4 or 5 strands wide, but 10 or more strands are possible. The arrangements may be purely parallel, purely anti-parallel, or mixed refer to Figure The hairpin bend is simply a bend in the primary structure held in place by a hydrogen bond.
Both are found on the exterior of proteins. Nonpolar side chains are hydrophobic and, although repelled by water, are attracted to each other.
Polar side chains attract other polar side chains through either dipole-dipole forces or hydrogen bonds. For example, both aspartic acid and glutamic acid yield side chains with a negative charge that are strongly attracted to the positive charges in the side chains of lysine and arginine. Two cysteine residues can connect by forming a disulfide linkage — a covalent bond Figure Examination of the structures of many proteins shows a preponderance of nonpolar side chains in the interior with a large number of polar or ionic side chains on the exterior.
In an aqueous environment, the hydrophobic nonpolar groups induce the protein to fold upon itself, burying the hydrophobic groups away from the water and leaving the hydrophilic groups adjacent to water. The result is similar in structure to a micelle. These interactions include hydrogen bonding and disulfide bonds. This quaternary structure locks the complex of proteins into a specific geometry.
An example is hemoglobin, which has four polypeptide chains. Dissecting a Protein for Study The previous sections have discussed the different types of protein structure.
Now it is time to see how a biochemist goes about determining the structure s of a particular protein. An animal generates an antibody in response to a foreign substance known as an antigen. Antibodies are proteins found in the blood serum. Exposure to diseases, certain chemicals, and allergies induce the formation of specific antibodies. These antigens collect on the surface of red blood cells.
Every antigen has a specific antibody. Antibodies are very specific and have a strong affinity for their specific antigens, recognizing specific amino acid sequences on the antigens. Animals have a large number of antibodies present in their bodies, based on their environmental history.
Separating proteins within a cell and purifying them There are thousands of different proteins in each cell. In order to examine and study one of them, you need to separate it from all the others.
The methods of separating proteins are, in general, applicable to all other types of biochemicals. Initially, simple filtration and solubility can remove gross impurities, but much more needs to be done before the sample is pure. The key separation and purification methods depend on two physical properties of the proteins: size and charge.
Separating proteins by size Methods relying on separation by protein size and mass include ultrafiltration, ultracentrifugation, and size exclusion chromatography. Ultrafiltration can separate smaller molecules from larger impurities or larger molecules from smaller impurities.
In ultracentrifugation, a powerful centrifuge causes heavier molecules to sink faster and, which allows their separation — much as the lighter water is separated from the heavier lettuce in a salad spinner. Ultracentrifugation also gives the molar mass of the protein.
In size exclusion chromatography, also known as molecular sieve chromatography or gel filtration chromatography, a solution passes through a chromatography column filled with porous beads. Molecules that are too large for the pores pass straight through. Molecules that may enter the pores are slowed. The molecules that may enter the pores undergo separation depending on how easily they can enter.
One of them is the examination of bloodstains, blood being the most common form of evidence examined by a forensic serologist. The presence of blood can link a suspect to both a victim and a crime scene.
Bloodstain patterns can also give evidence of how a violent attack took place. Criminals recognize the significance of this evidence and often try to conceal it. The luminol test is useful in detecting invisible bloodstains because, in contact with blood, or a few other chemicals, luminol emits light, which can be seen in a darkened room. The Wagenhaar, Takayama, and Teichman tests take advantage of the fact that long-dried blood will crystallize or can be induced to crystallize.
Blood is mostly water, but it also contains a number of additional materials including cells, proteins, and enzymes. The fluid portion, or plasma, is mostly water. The serum is yellowish and contains platelets and white blood cells.
The platelets, or red blood cells, outnumber the white blood cells by about to 1. White blood cells are medically important, whereas red blood cells and, to a lesser extent, serum are important to the forensic serologist.
Because blood quickly clots when exposed to air, serologists must separate the serum from the clotted material. The serum contains antibodies that have forensic applications, and red blood cells have substances such as antigens on their surfaces that also have forensic applications.
Antibodies and antigens are the keys to forensic serology: Even identical twins with identical DNA have different antibodies. As you know from this chapter, antibodies, and some antigens, are proteins, and this is why methods of studying proteins are important to their analysis. The forensic investigator answers this question and the next one, if applicable by means of an antiserum test.
It is important to know whether the blood came from a human or an animal such as a pet. The standard test is the precipitin test. If human antiserum creates clotting in a blood sample, the sample must be human. Analysis of bloodstains initially attempts to answer five questions. To answer this question, the investigator can use a number of tests. The generic term for a test of this type is a presumptive test.
It is possible to create animal antiserums in an analogous manner, and test for each type of animal. The procedure for answering this depends on the quantity and quality of the sample.
If the quality is good, direct typing is done — otherwise, indirect typing is used. A dried bloodstain normally requires indirect typing. The most common indirect typing method is the absorptionelution test. Treatment of a sample with antiserum antibodies gives a solution which, upon addition to a known sample, causes coagulation. Here the answers become less precise.
Clotting and crystallization indicate age. Testing for testosterone levels and chromosome testing can determine sex. And certain controversial, racial genetic markers based on protein and enzyme tests may indicate race.
Other body fluids may contain the same antibodies and antigens found in blood. Therefore, similar tests work on these fluids as well. Separating proteins by charge Methods of separating proteins relying on the charge of the protein include solubility, ion exchange chromatography, and electrophoresis.
Each of these methods is pH dependent. Proteins are least soluble at their isoelectric point. The isoelectric point is the pH where the net charge on the protein is 0. At the isoelectric point, many proteins precipitate from solution. At a pH below the isoelectric point, the protein has a net positive charge, whereas a pH above the isoelectric point imparts a net negative charge.
The magnitude of the charge depends on the pH and the identity of the protein. Therefore, two proteins coincidently having the same isoelectric point will not necessarily have the same net charge at a pH that is one unit lower than the isoelectric point.
Both ion exchange chromatography and electrophoresis take advantage of the net charge. In ion exchange chromatography, the greater the magnitude of the charge, the slower a protein moves through a column — this is similar to the ion-exchange process that occurs in water-softening units. In electrophoresis, the sample solution is placed in an electrostatic field. Molecules with no net charge do not move, but species with a net positive charge move toward the negative end, and those with a net negative charge move toward the positive end.
The magnitude of the net charge determines how fast the species moves. Other factors influence the rate of movement, but the charge is the key. There are numerous modifications of electrophoresis. In protein analysis, rarely do biochemists use only one single technique.
They commonly use several in order to confirm their findings. Because many proteins only have one polypeptide chain, this step is not always necessary. Denaturing the protein, disrupting its threedimensional structure without breaking the peptide bonds, using pH extremes will normally suffice. If disulfide linkages are present between the chains, apply the procedure outlined in Step 2 to separate the chains for isolation. Step 2: Slashing intrachain disulfide linkages Step 2 requires breaking cleaving the disulfide linkages.
A simple reduction accomplishes this. However, the linkages may reform later, so it is necessary to cleave the linkages and prevent their reformation via reductive cleavage followed by alkylation.
Oxidative cleavage, where oxidation of the sulfur to —SO3— occurs, also prevents a reversal of the process. Step 3: Determining amino acid concentration of the chain Step 3 is easily accomplished using an amino acid analyzer, an automated instrument that can determine the amino acid composition of a protein in less than an hour.
The instrument requires less than a nanomole of protein. Step 4: Identifying the terminal amino acids Step 4 not only identifies the terminal amino acids but also indicates whether more than one chain is present. A polypeptide chain only has one N-terminal and one C-terminal amino acid. Therefore, if more than one N- or C-terminal amino acid is present, there must be more than one polypeptide chain.
It is possible to identify the N-terminal residue in a number of ways. In general, procedures begin by adding a reagent that reacts with the N-terminal amino acid and tags it.
Subsequent hydrolysis destroys the polypeptide, allowing separation of the tagged residue and its identification. The method of choice nowadays is called the Edman degradation. This method, as do other methods, tags the N-terminal residue; however, only the terminal amino acid is cleaved from the chain, so the remainder of the chain is not destroyed as in other methods. It is possible to repeat the procedure on the shortened chain to determine the next residue.
In principle, repetition of the Edman degradation can yield the entire sequence, but, in most cases, determination of the first 30 to 60 residues is the limit. The akabori reaction hydrazinolysis and reduction with lithium aluminum hydride tag the C-terminal residue. It is also possible to selectively cleave the C-terminal residue using the enzyme carboxypeptidase, a variety of which are available.
Steps 5 and 6: Breaking the chain into smaller pieces In Step 5, you cleave the polypeptide into smaller fragments and determine the amino acid composition and sequence of each fragment. Step 6 repeats Step 5 using a different cleavage procedure to give a different set of fragments. Steps 5 and 6 break the chain into smaller pieces to ease identification.
Most of the methods here employ enzymes; however, other less-specific methods are useful in some cases. Partial acid hydrolysis randomly cleaves the protein chain into a number of fragments.
Trypsin, a digestive enzyme, specifically cleaves on the C-side of arginine or lysine. Using trypsin gives additional information that the total number of arginine and lysine residues present is one less than the number of fragments generated.
The digestive enzyme chymotrypsin preferentially cleaves residues containing aromatic rings tyrosine, phenylalanine, and tryptophan. It slowly cleaves other residues especially leucine. Clostripain cleaves positively charged amino acids, especially arginine. It cleaves lysine more slowly. Fragments with a C-terminal aspartic acid or glutamic acid form from the interaction of staphylococcal protease on a protein in a phosphate buffer. In the presence of bicarbonate or acetate buffer, only C-terminal glutamic acid fragments result.
A number of less specific enzymes can complete the breakdown of the fragments, including elastase, subtilisin, thermolysin, pepsin, and papain. Chemical methods of breaking up the fragments include treatment with cyanogen bromide, hydroxylamine, and heating an acidic solution.
Cyanogen bromide specifically attacks methionine. Hydroxylamine specifically attacks asparagine-glycine bonds. It is possible to apply the Edman degradation on each of the fragments. This can simplify the determination of the sequence of a large protein. Step 7: Combining information to get the total sequence Step 7 is where the information from the various procedures comes together. For example, look at a simple octapeptide fragment from a protein. This fragment gave, upon complete hydrolysis, one molecule each of alanine Ala , aspartic acid Asp , glycine Gly , lysine Lys , phenylalanine Phe , and valine Val , and two molecules of cysteine Cys.
Over the years, additional reactions have been discovered. More than antigens are known, leading to 23 different blood groups. Each blood group is defined by the antibodies present in the serum and the antigens present on the red blood cells. In basic blood typing, one needs two antiserums, labeled anti-A and anti-B. Adding a drop of one of these to a blood sample causes coagulation if the appropriate antigens are present.
Anti-A interacts with both A and AB blood. Anti-B interacts with both B and AB blood. Neither interacts with type O blood. The approximate distribution of the different blood types is: 43—45 percent type O; 40—42 percent type A; 10—12 percent B; and 3—5 percent AB.
Subgrouping is also possible with designations such as O1 and O2. There are other very rare types as well. The Rh factor provides an additional means of subdividing blood.
The Rh factor the name comes from the rhesus monkey is an antigen on the surface of red blood cells. A person with a positive Rh factor contains a protein antibody that is also present in the bloodstream of the rhesus monkey.
About 85 percent humans are Rh positive. A person lacking this protein is, naturally, Rh negative. Assigning a blood sample as Rh positive or Rh negative is a useful simplification.
There are about 30 possible combinations of factors. Additional factors can determine whether blood belongs to a specific individual: the identification of other proteins and enzymes present in the blood. One of the characteristics of proteins or enzymes in the blood is polymorphism, or the ability to be present as isoenzymes.
Polymorphism means that the protein may exist in different forms or variants. One well-known example is the polymorphism of hemoglobin into the form causing sickle cell anemia.
The determination of each of these additional factors narrows down the number of possible individuals. If the disulfide linkages are left intact by skipping Step 2, different fragments result. This can be used to determine the overall shape of a protein. In some cases, more detailed structural information can be determined by sophisticated instrumental analysis techniques.
As catalysts, they alter the rate of a chemical reaction without themselves being consumed in the reaction. Enzymes are normally very specific in their action, often targeting only one specific reacting species, known as the substrate. This specificity includes stereospecificity, the arrangement of the substrate atoms in three-dimensional space.
Stereospecificity is illustrated by the fact that if the D-glucose in your diet were replaced by its enantiomer, L-glucose, you would not be able to metabolize this otherwise identical enantiomer. Enzymes occur in many forms. Some enzymes consist entirely of proteins, whereas others have non-protein portions known as cofactors. The cofactor may be a metal ion, such as magnesium, or an organic substance.
We call an organic cofactor a coenzyme there is no specific term for a metallic cofactor. An enzyme lacking its cofactor is an apoenzyme, and the combination of an apoenzyme and its cofactor is a holoenzyme.
A metalloenzyme contains an apoenzyme and a metal ion cofactor. A tightly bound coenzyme is a prosthetic group. We know that this is a lot of terminology, but hang in there. The key is the enzyme. One region on the enzyme, the active site, is directly responsible for interacting with the reacting molecule s. When a reacting molecule, the substrate, binds to this active site, a reaction may occur.
Other materials besides the enzyme and substrate, may be necessary for the reaction to occur. The enzyme trypsin illustrates why it is sometimes necessary to generate an inactive form of an enzyme. Trypsin is one of the enzymes present in the stomach that is responsible for the digestion of proteins.
Its production, as an inactive form, occurs in the cells of the stomach walls, and activation occurs after its release into the stomach. If trypsin were produced in the active form, it would immediately proceed to begin digesting the cell that produced it.
Eating yourself is not a good thing. The activation of the inactive form of an enzyme serves as one form of enzyme control. Inhibition is another method of enzyme control. The two general types of inhibition are competitive inhibition and noncompetitive inhibition. In competitive inhibition, another species competes with the substrate to interact with the active site on the enzyme.
In noncompetitive inhibition, the other species binds to some site other than the active site. This binding alters the overall structure of the enzyme so that it no longer functions as a catalyst. Common names for enzymes begin with some description of its action plus an -ase suffix. Enzymes that were named before the implementation of the -ase system, such as trypsin, do not follow this convention. The Enzyme Commission has also developed a numerical system for classifying enzymes.
The names begin with EC, for Enzyme Commission, and end with four numbers, separated by decimal points, describing the enzyme. An example of this nomenclature is EC 2. The first number in the EC name refers to the major enzyme class, and there are six major enzyme classes, summarized in Table To continue with our example, the 2 in EC 2. The second number, the 7, indicates what group the enzyme transfers. The third number, the first 4, indicates the destination of the transferred group.
And the last number, the second 4, refines the information given by the third number. An oxidation involves the increase in the oxidation state of an element, whereas a reduction involves the decrease in the oxidation state of an element.
It is impossible to have one without the other. Examples of the types of reactions that qualify as oxidation and reduction reactions are in Table In general, the substrate undergoes either oxidation or reduction, while the enzyme temporarily does the opposite but eventually returns to its original form.
Table Some Possible Types of Oxidation and Reduction Reactions Oxidation Reduction Loss of one or more electrons Gain of one or more electrons Addition of oxygen Loss of oxygen Loss of hydrogen Gain of hydrogen An example: Succinate dehydrogenase catalyzes the oxidation of the succinate ion.
In this case, the oxidation involves the loss of two hydrogen atoms with the formation of a trans double bond. Aminotransferase transfers an amino group, and phosphotransferase transfers a phosphoryl group. The general form, unbalanced, of these reactions appears in Figure There may be a pH dependence, which results in the subsequent loss of a hydrogen ion.
A phosphatase catalyzes the hydrolysis of a monophosphate ester, and a peptidase catalyzes the hydrolysis of a peptide bond. The general form of these reactions appears in Figure This process is accompanied by the formation of a double bond or the addition of a group to a double bond.
A deaminase aids in the removal of ammonia, and a decarboxylase catalyzes the loss of CO2. Isomerase enzymes catalyze the conversion of one isomer to another.
The racemase illustrated at the top of Figure catalyzes the racemization of enantiomers. An epimerase, like the one at the bottom of Figure , catalizes the change of one epimer to another. Like all catalyzed reactions, these are equilibrium processes. Putting it together: Ligases Ligase enzymes catalyze reactions leading to the joining of two molecules in which a covalent bond forms between the two molecules. The process often utilizes high-energy bonds such as in ATP.
Figure illustrates the action of two ligases, pyruvate carboxylase and acetyl-CoA synthetase. Pyruvate carboxylase catalyzes the formation of a C-C bond. Acetyl-CoA synthetase catalyzes the formation of a C-S bond. In this formation, the substrate in some way binds to the active site of the enzyme. The interaction between the enzyme and the substrate must, in some way, facilitate the reaction, and it opens a new reaction pathway.
The active site is typically a very small part of the overall enzyme structure. The amino acid residues comprising the active site may come from widely separated regions of the protein primary structure , and it is only through interactions leading to higher structure levels that they are brought close together.
Amino acid residues not in the active site serve many different functions that aid the function of the enzyme. Models of catalysis: Lock and key versus induced-fit The first attempt at explaining this process led to the Lock and Key Model, in which the substrate behaves as a key that fits into a lock, the enzyme Figure The Lock and Key Model, to a certain degree, explains the specificity of enzymes.
Just as only the right key will fit into a lock, only the right substrate fits into the enzyme. The modification begins the process of the reaction. Figure illustrates how the Induced-Fit Model applies to the formation of the same enzyme-substrate in Figure All About Kinetics As you know, all reactions involve energy.
If the energy level of the products is greater than that of the reactants energy is absorbed , the reaction is endergonic, and nonspontaneous.
If the energy level of the products is less than the reactants energy is released , the process is exergonic, and spontaneous. The difference between the reactants and products remains unchanged, as does the equilibrium distribution of the reactants and products.
The enzyme facilitates the formation of the transition state Figure These two fates lead to two equilibria. One of the equilibria involves the reactant substrate and the transition state, and the other involves the product s and the transition state.
Rapid removal of the product s does not allow establishment of the reverse process that leads to the equilibrium. Removal of the product simplifies the analysis of the kinetic data. Enzymes, like all catalysts, catalyze both the forward and the reverse reaction.
The ultimate equilibrium concentrations of substrate and products will be the same whether an enzyme is present or not — the enzyme merely changes the amount of time necessary to reach this state. Enzyme assays: Fixed time and kinetic An enzyme assay is an experiment to determine the catalytic activity of an enzyme.
It is possible to measure either the rate of disappearance of the substrate or the rate of appearance of a product. The experimental mode of detection depends on the particular chemical and physical properties of the substrate or the product, and the rate is the change in concentration per change in time. In fixed time assay, you simply measure the amount of reaction in a fixed amount of time.
In kinetic assay, you monitor the progress of a reaction continuously. Once you determine the rate of change in concentration of any reactant or product, it is possible to determine the rate of change of for any other reactant or product of the reaction It is important to control the conditions precisely.
Minor changes in variables such as the temperature or the pH can drastically alter the catalytic activity of an enzyme. Rate determination: How fast is fast? It is important to control kinetic experiments closely.
Once you determine the basic conditions, you can run a series of experiments using a fixed enzyme concentration and varying concentrations of substrate. Up to a point, an increase in substrate concentration results in an increase in rate. The rate increases until the enzyme is saturated. This saturation point is where all the enzyme molecules are part of an enzyme-substrate complex. For examine, creatine kinese CK is an enzyme that aids in the synthesis and degradation of creatine phosphate.
CK exists as three different isoenzymes. Each is composed of two polypeptide chains. CK found in the brain also has identical polypeptide chains, but they are different from the ones associated with muscle CK and are labeled CK-BB.
When tissue undergoes injury, though, some of the intracellular enzymes leak into the blood where they can be measured. Elevated levels of total CK all three isoenzymes may be indicative of sketalmuscle trauma or myocardial infarction MI, or heart attack. Analysis of the individual isoenzymes may give additional clues.
For example, an individual falls off a ladder and suffers several broken bones. He is taken to the hospital, where his blood serum CK is measured.
It is elevated as expected, but the physician also orders a CK-MB level determination. It turns out to also be highly elevated, indicating that the reason the man fell off the latter to begin with was that he was suffering a heart attack CK-MB. This knowledge allows the doctor to start a regime of treatment that helps to minimize permanent heart damage. For most reactions, the rate of the reaction approaches the saturation level along a hyperbolic curve.
Theoretically, the reaction rate will only reach saturation at infinite substrate concentration. A plot of the reaction rate, V, versus the substrate concentration, [substrate], supplies several bits of useful data see Figure The experiment is at constant enzyme concentration. One piece of useful data is the maximum reaction rate, Vmax. The rate approaches Vmax asymptotically. At low substrate concentrations, the reaction approaches first-order kinetics, where the rate of reaction depends only on the concentration of one reactant.
At high concentrations, the reaction approaches zero-order kinetics, where the rate of reaction is independent of reactant concentration. Later in this chapter you will see that this graph varies with less simple enzyme-substrate interactions. In the region between the zero-order region and the first-order region, the kinetics are mixed and difficult to interpret.
The Michaelis constant, measured in terms of molarity, is a rough measure of the enzymesubstrate affinity. KM values vary widely. At high substrate concentrations, though, V is nearly independent of [substrate]. The low substrate region is useful in the application of the Michaelis-Menten equation see the next section.
In an uncatalyzed reaction, increasing the substrate concentration does not lead to a limiting Vmax. The rate continues to increase with increasing substrate concentration. This indirect evidence leads to the conclusion that there is an enzyme-substrate complex, a tightly-bound grouping of the enzyme and the substrate.
The limit occurs when all the enzyme molecules are part of a complex so that there are no free enzyme molecules available to accommodate the additional substrate molecules. Various x-ray and spectroscopic techniques provide direct evidence to confirm the formation of an enzymesubstrate complex. It is possible to interpret the behavior of many enzymes by applying the equation to kinetic data. In general, the results of the kinetics experiments are for allosteric enzymes. As seen in Figure , the rate of catalysis, V, increases linearly at low substrate concentration, but begins to level off at higher concentrations.
The various instances of k refer to the rate constants of the various steps — a negative rate constant is for the reverse process. In the first step, the separate enzyme and substrate combine to form the enzyme-substrate complex transition state. The rate of formation of ES is k1. After ES forms, it may break down to E and S k-1 or it may proceed to product k2. Note: Some texts refer to k2 as kcat. Because the enzyme will catalyze the reverse process, E and P may combine to reform the complex k Ignoring the reverse reaction k-2 simplifies the interpretation of the data.
This is not an unreasonable assumption if data collection is near the beginning of the reaction, where the concentration of P is low. Through their work, an expression relating the catalytic rate to the concentrations of the enzyme and substrate and to the individual rates was developed. Throughout most of the reaction, the concentration of ES remains nearly constant.
This is the steady-state assumption, which assumes that during a reaction the concentrations of any intermediates remain nearly constant.
The value of Vmax supplies the turnover number of the enzyme. The turnover number gives the number of substrate molecules transforming to products per unit of time for a fully saturated enzyme. You can determine k2 from this value. The constant k2 is also known as the catalytic constant, kcat. In cells, however, the enzymes are seldom saturated with substrate.
If KM is much greater than [S], the catalytic rate kcat or k2 is significantly less than the ideal value because only a small portion of the active sites contain substrate. The maximum rate of catalytic activity is limited by the rate of diffusion to bring the enzyme and substrate together.
Some enzymes can exceed this limit by forming assemblages. In these groups, the product of one enzyme is the substrate for a closely associated enzyme. This allows a substrate to enter the group and pass from enzyme to enzyme as if it were in an assembly line. Another complication is that many enzymes require more than one substrate. It is possible to utilize these multiple substrates through sequential displacement or through double displacement.
In sequential displacement, all substrates must simultaneously bind to the enzyme before the release of the product. In this type of displacement, the order in which the substrates bind is unimportant. In double displacement, or ping-pong, situations, one or more products leave before all the substrates bind. Double displacement mechanisms temporarily modify the enzyme.
Here we go again: Lineweaver-Burk plots Once upon a time, before the invention of computers, the determination of KM and Vmax was a tedious process.
Today curve-fitting programs allow rapid analysis of the data to determine these values. From water biochemistry to protein synthesis, Biochemistry For Dummies gives you the vital information, clear explanations, and important insights you need to increase your understanding and improve your performance on any biochemistry test. If the link is not responding kindly inform us through comment section.
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