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1.1
Enzymes as Biocatalysts
1.1.1
An Overview
There are two fundamental conditions for life. First, the living entity must be able to
self-replicate; second, the organism must be able to catalyze chemical reactions
efficiently and selectively. The living systems make use of energy from the environment.
Many of us, for example, consume substantial amounts of sucrosecommon table
sugaras a kind of fuel, whether in the form of sweetened foods and drinks or as sugar
itself. The conversion of sucrose to CO2 and H2O in the presence of oxygen is a highly
exergonic process, releasing free energy that we can use to think, move, taste, and see.
However, a bag of sugar can remain on the shelf for years without any obvious
conversion to CO2 and H2O. Although this chemical process is thermodynamically
favorable, it is very slow! Yet when sucrose is consumed by a human (or almost any
other organism), it releases its chemical energy in seconds.
The difference is catalysis. Without catalysis, chemical reactions such as sucrose
oxidation could not occur on a useful time scale, and thus could not sustain life. Almost
all enzymes are proteins. However, there are also catalytically active ribonucleic acids,
the ribozymes. Enzymes have extraordinary catalytic power, often far greater than that
of synthetic or inorganic catalysts.
They have a high degree of specificity for their substrates, they accelerate chemical
reactions tremendously, and they function in aqueous solutions under very mild
conditions of temperature and pH. Few nonbiological catalysts have all these properties.
Enzymes are central to every biochemical process. Acting in organized sequences, they
catalyze the hundreds of stepwise reactions that degrade nutrient molecules, conserve
and transform chemical energy, and make biological macromolecules from simple
precursors. Through the action of regulatory enzymes, metabolic pathways are highly
1.1.2
A Brief History
notions of life were disproved, the isolation of new enzymes and the investigation of
their properties advanced the science of biochemistry.
The isolation and crystallization of urease by James Sumner in 1926 provided a
breakthrough in early enzyme studies. Sumner found that urease crystals consisted
entirely of protein, and he postulated that all enzymes are proteins. In the absence of
other examples, this idea remained controversial for some time. Only in the 1930s was
Sumners conclusion widely accepted, after John Northrop and Moses Kunitz
crystallized pepsin, trypsin, and other digestive enzymes and found them also to be
proteins. During this period, JBS Haldane wrote a treatise entitled Enzymes. Although
the molecular nature of enzymes was not yet fully appreciated, Haldane made the
remarkable suggestion that weak bonding interactions between an enzyme and its
substrate might be used to catalyze a reaction. This insight lies at the heart of our
current understanding of enzymatic catalysis.
Since the latter part of the twentieth century, research on enzymes has been intensive. It
has led to the purification of thousands of enzymes, elucidation of the structure and
chemical mechanism of many of them, and a general understanding of how enzymes
work.
1.2
1.2.1
How
are
Enzymes
Important
in
Living
Organisms?
Enzymes are organic catalysts which aid in facilitating chemical reactions in the body.
Enzymes are substances which make life possible and which are found in natural, live
foods and also in your body. Enzymes are the work force of the body. Without them,
chemical reactions cannot take place, and hormones, minerals, and vitamins cannot
carry out their functions. There are believed to be hundreds of thousands of enzymes in
the body; different enzymes perform different functions. Without them, life cannot exist.
Some activities of enzymes are:
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Work to store food in the liver and muscles for fuel later on
Coagulate blood
Promote oxidation
You can have all the raw materials necessary for good health vitamins, minerals,
intrinsic factors, proteins, carbohydrates, fats, amino acids, etc. but enzymes are
necessary in order for your body to utilize all the raw materials in its life-supporting
activities of metabolism.
Vitamins, minerals and all kinds of super-nutrients are in the spotlight on the
nutritional arena. Enzymes are not that much talked about though. But they are
essential and most of the people these days, including small children are very deficient!
Actually, we are the only species on Earth that tries to live without food enzymes! And
were doing a poor job at it.What happened and why are enzymes so necessary for
health?
Our bodies are designed to rely on two sources of enzymes in digesting food the enzymes
produced by our digestive system and the enzymes naturally present in the food we eat.
Raw food like uncooked fruits and vegetables contribute enzymes to the digestive
process, but each one brings only enough enzymes to digest itself and no more.
Whenever food is cooked or processed at any time before it is eaten whether it is
microwaved, steamed, broiled, roasted, pasteurized, canned, sauted, stir-fried or heated
in any manner all of the food enzymes are destroyed in a few short minutes. And this
doesn't mean you have to do the cooking the enzymes in all processed foods, whether
you cook them or not, have been destroyed. In his book, Enzyme Nutrition, Dr. Edward
Howell observed that the refining of food and improved cooking methods have rendered
the modern diet enzyme-deficient due to the effective destruction of enzymes in foods by
these processes.
The critical role of enzymes in the maintenance of health and well-being was
dramatically demonstrated in an experiment now known as The Pottenger Cat
Studies, which was performed by Francis Pottenger in 1946. Pottenger wanted to
know if raw meat and raw milk, when modified by high heat, had an impact on growth
and development. During the ten years of the study, nine hundred cats were studied.
The results of this study were stunning: consuming raw meat and raw milk versus
heated and cooked meat and milk can negatively affect cats health over four
generations!! After just one generation of cats, which was fed cooked food, these
developed degenerative diseases.
Digestive enzymes, which digest the food; most are manufactured by the pancreas
Food enzymes obtained from raw foods, which start food digestion
Our bodies are run by metabolic enzymes; every organ and tissue has its own particular
metabolic enzymes to do specialized work. Since good health depends on all of these
metabolic enzymes doing an excellent job, we must be sure that nothing interferes with
the body making enough of them. A shortage could mean trouble, many-time serious.
Natures plan calls for food enzymes to carry the whole load. If food enzymes do some of
the work, the enzyme potential can have much more to give to the hundreds of
metabolic enzymes that run the body.
So, when ingested, the enzymes in raw food or supplementary enzymes, result in a
significant degree of digestion, lowering the drain on the organism. The heat in cooking
destroys enzymes and forces the organism to produce more enzymes, thus enlarging the
pancreas. This way, the body is unable to produce an adequate quantity of metabolic
enzymes to repair the body and fight disease.
1.2.3 Health
Conditions
Associated
With
Enzyme
Deficiency
According to Dr. Edward Howell, a noted pioneer in the field of enzyme research,
enzyme deficiency leads to a shortened lifespan, illness and lowered resistance to illness.
On short, the less your supply of enzymes is, the shorter your life! Thats a pretty good
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argument and a very truthful one as well! The reality is just this: a body in a weakened,
enzyme-deficient state is a prime target for cancer, obesity, arthritis, allergies, heart
disease and other degenerative problems. The glands and the major organs, including
the brain, suffer most from the unnatural digestive drain on the metabolic enzyme
potential. The pancreas swells to meet the great demand for its juices while other glands
also abnormally adapt, and the brain actually shrinks on the all cooked and over-refined
diet.
Enzymes are needed for all metabolic pathways in the body, respiration, digestion and
other important life processes. When enzymes function properly, homeostasis is
maintained. However, if an enzyme is lacking or has an incorrect shape due to a genetic
mutation, this can lead to disease within an organism. An example of this is the disease
phenylketonuria or PKU. It is an autosomal recessive disorder which involves a gene
mutation in the gene for the enzyme phenylalanine hydroxylase (PAH). This enzyme is
part of a metabolic pathway in which the amino acid phenylalanine is converted to
tyrosine. In the person with PKU disease, they have an enzyme that is defective and the
activity is reduced. Therefore, phenylalanine accumulates and is converted to
phenylpyruvate, which is found in the urine. It also builds up in the brain, leading to
mental retardation and brain damage. By restricting the person's intake of protein,
which contains phenylalanine, the person can have a normal life span and mental
development. This is but one example of the many enzymes which have very necessary
and specific functions in the body.
Medical scientists address imbalances in enzyme activity by using pharmacologic agents
to inhibit specific enzymes and are investigating gene therapy as a means to remedy
deficits in enzyme level or function.
normally active in the blood for lack of their specific substrates, but their enzymatic
activity can be measured in a test tube by the addition of the appropriate substrates to
samples of plasma. Such measurements are clinically useful because abnormally high
plasma concentrations of particular enzymes are characteristic of certain diseases
(Table 1.1).
For example, alanine transaminase (ALT) or serum glutamic-pyruvic transaminase
(SGPT) is an enzyme that helps to process proteins. Large amounts of ALT occur in liver
cells. When the liver is injured or inflamed (as in hepatitis), the blood level of ALT
usually
rises.
Aspartate
aminotransferase
(AST)
or
serum
glutamic-
oxaloacetic transaminase (SGOT) is an enzyme usually found inside liver cells. When a
blood test detects high levels of this enzyme in the blood it usually means the liver is
injured in some way. However AST can also be released if heart or skeletal muscle is
damaged. For this reason ALT is usually considered to be more specifically related to
liver problems. Alkaline phosphatase (ALP) is another enzyme occurs mainly in liver
cells next to bile ducts, and in bone. The blood level is raised in some types of liver and
bone disease.
Table 1.1: Examples of the diagnostic value of some enzymes found in plasma
1.3
Commercially useful enzymes (CUEs) are enzymes which have commercial uses.
Microbial enzymes have well-known applications as biocatalysts in several areas of
industry, such as biotechnology, agriculture, pharmaceuticals, etc. Metagenomic data
provide a unique resource for discovering novel commercially useful enzymes (CUEs)
from yet unidentified microbes belonging to complex microbial communities in diverse
ecosystems.
1.3.1
compared
with
chemical
processes,
enzyme-based
processes
are
Several thousand tonnes of commercial enzymes are currently produced each year,
which have a value in excess of US$1500 million. A few animal and plant enzymes are
used, but most commercial enzymes are now obtained from microbial sources. Many of
these are extracellular enzymes, with the majority being derived from various species of
Bacillus. Their proteases and amylases are the most widely used, and there is a
particular demand for the thermostable enzymes that are available from several
members of this genus. The greatest proportion of all commercial enzymes, some 34%,
are used as detergent enzymes, 14% for dairy-related uses, with 12% for starch
processing and 11% for textile applications. The remaining 29% are divided among a
vast array of applications (Figure 1.1 and Table 1.2).
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Besides their role as aids in traditional processes, bulk enzymes are at the centre of
many novel processes and biotechnological innovation continues to expand the range of
applications. Bulk enzymes are normally quite crude preparations that are purified only
sufficiently to fulfil customer requirements for activity and stability. They often contain
many other enzymes, which in some instances are beneficial to overall application
performance.
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There is an increasing demand for fine enzymes used in molecular biology, particularly
restriction endonucleases and DNA polymerases, e.g., DNA polymerase from Thermus
aquaticus (Taq DNA polymerase) or Pyrococcus furiosus (Pfu DNA polymerase). These
enzymes are used in polymerase chain reaction (PCR) for DNA fragment amplification.
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1.4
Characteristics
of
Enzyme-Catalyzed
Reactions
1.4.1
Most of the chemical reactions in the body or in any living cell, if carried out in a test
tube with only reactants and products present, would proceed at very low rates because
they have high activation energies. In order to achieve the high reaction rates observed
in living organisms, catalysts are required to lower the activation energies. These
particular catalysts are called enzymes (meaning in yeast since the first enzymes were
discovered in yeast cells). Enzymes are protein molecules, so an enzyme can be defined
as a protein catalyst. (Although some RNA molecules possess catalytic activity, the
number of reactions they catalyze is very small, and we shall restrict the term enzyme
to protein catalysts.)
To function, an enzyme must come into contact with reactants, which are called
substrates in the case of enzyme-mediated reactions. The substrate becomes bound to
the enzyme, forming an enzyme-substrate complex, which breaks down to release
products and enzyme. The reaction between enzyme and substrate can be written:
At the end of the reaction, the enzyme is free to undergo the same reaction with
additional substrate molecules. The overall effect is to accelerate the conversion of
substrate into product, with the enzyme acting as a catalyst. Note that an enzyme
increases both the forward and reverse rates of a reaction and thus does not change the
chemical equilibrium that is finally reached.
The interaction between substrate and enzyme has all the characteristics for the binding
of a ligand to a binding site on a proteinspecificity, affinity, competition, and
saturation. The region of the enzyme to which the substrate binds is known as the
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enzymes active site (a term equivalent to binding site). The shape of the enzyme in
the region of the active site provides the basis for the enzymes chemical specificity since
the shape of the active site is complementary to the substrates shape (Figure 1.2).
There are approximately 4,000 different enzymes in a typical cell, each capable of
catalyzing a different chemical reaction. Enzymes are generally named by adding the
suffix -ase to the name of either the substrate or the type of reaction catalyzed by the
enzyme.
Figure 1.2: Binding of substrate to the active site of an enzyme catalyzes the formation
of products.
1.4.2 Enzymes
Have
High
Specificity
and
Rapid
Reaction Rates
The twin phenomenon of high specificity and rapid reaction rates are the cornerstones
of enzyme activity.
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molecular size, shape, and charge distribution of both the enzyme and substrate must be
compatible for this selective binding process to occur.
Let us consider proteolytic enzymes as an example (Figure 1.3). In vivo, these enzymes
catalyze proteolysis, the hydrolysis of a peptide bond.
Figure 1.4: Enzyme specificity. (a) Trypsin cleaves on the carboxyl side of arginine and
lysine residues, whereas (b) thrombin cleaves Arg-Gly bonds in particular sequences
specifically.
Enzymes are also stereospecific catalysts and typically catalyze reactions only of specific
stereoisomers of a given compoundfor example, D- but not L-sugars, L- but not D15
amino acids. Since they bind substrates through at least three points of attachment,
enzymes can even convert nonchiral substrates to chiral products. Figure 1.5 illustrates
why the enzyme-catalyzed reduction of the nonchiral substrate pyruvate produces Llactate rather a racemic mixture of D- and L-lactate.
1.4.2.2
The enzyme transforms the substrate into the product with lightning speed. In fact,
enzyme-catalyzed reactions often occur from one million to 100 million times faster that
the corresponding uncatalyzed reaction. Like all catalysts, enzymes are neither
consumed nor permanently altered as a consequence of their participation in a reaction.
Catalase: The catalase enzyme provides one of the most spectacular examples of the
increase in reaction rates brought about by enzymes (Figure 1.6). This enzyme is
required for life in an (oxygen-requiring) breakdown of food molecules produces
hydrogen peroxide (H2O2). Because H2O2 is toxic to the cell, it must be destroyed. One
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Figure 1.6: The enzyme catalase catalyzes the decomposition of hydrogen peroxide
(H2O2) to water and oxygen.
This is the same reaction that you witness when you pour hydrogen peroxide on a
wound. The catalase released from injured cells rapidly breaks down the hydrogen
peroxide. The bubbles that you see are oxygen gas released as a product of the reaction.
Carbonic Anhydrase: Another example, the reaction in which carbonic acid is
broken down into carbon dioxide and water is catalyzed by the enzyme carbonic
anhydrase (Figure 1.7). The transfer of CO2 from the tissues into the blood and then to
the alveolar air would be less complete in the absence of this enzyme. In fact, carbonic
anhydrase is one of the fastest enzymes known. Each enzyme molecule can hydrate 106
molecules of CO2 per second. This catalyzed reaction is 107 times as fast as the
uncatalyzed one.
Figure 1.7: Carbon dioxide is a major end product of aerobic metabolism. In complex
organisms, this carbon dioxide is released into the blood and transported to the lungs
for exhalation. While in the blood, carbon dioxide reacts with water to form carbonic
acid, a hydration reaction catalyzed by carbonic anhydrase. The product of this reaction
is a moderately strong acid, carbonic acid (pKa = 3.5), which becomes bicarbonate ion
on the loss of a proton.
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1.5
1.5.1
In order for a given reaction to occur, the reactants must have sufficient energy. The
amount of energy required for a reaction to proceed is called the activation energy
(Figure 1.8). By analogy, a match will not burn and release heat energy unless it is first
activated by striking the match or by placing it in a flame.
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Figure 1.8: A comparison of noncatalyzed and catalyzed reactions. The upper figures
compare the proportion of reactant molecules that have sufficient activation energy to
participate in the reaction (blue = insufficient energy; green = sufficient energy). This
proportion is increased in the enzyme-catalyzed reaction because enzymes lower the
activation energy required for the reaction (shown as a barrier on top of an energy hill
in the lower figures). Reactants that can overcome this barrier are able to participate in
the reaction, as shown by arrows pointing to the bottom of the energy hill.
In a large population of molecules, only a small fraction will possess sufficient energy for
a reaction. Adding heat will raise the energy level of all the reactant molecules, thus
increasing the percentage of the population that has the activation energy. Heat makes
reactions go faster, but it also produces undesirable side effects in cells. Catalysts make
reactions go faster at lower temperatures by lowering the activation energy required,
thus ensuring that a larger percentage of the population of reactant molecules will have
sufficient energy to participate in the reaction (Figure 1.8).
19
Since a small fraction of the reactants will have the activation energy required for a
reaction even in the absence of a catalyst, the reaction could theoretically occur
spontaneously at a slow rate. This rate, however, would be much too slow for the needs
of a cell. So, from a biological standpoint, the presence or absence of a specific enzyme
catalyst acts as a switchthe reaction will occur if the enzyme is present and will not
occur if the enzyme is absent.
1.5.2
The activation energy is a term coined by the Swedish scientist, Svante Arrhenius in
1889. It means the amount of energy expressed in joules that is required to convert the
molecules in one mole of a reactant from a ground state to the transition state (Figure
1.9). It can also mean the energy that an atomic system must have before an emission or
chemical reaction can occur.
Figure 1.9: Activation energy of a reaction. Most reactions in a cell require very high
temperatures to get going, which would destroy the cell. Enzymes work by lowering the
activation energy of a reaction. The activation energy of a reaction is lowered by putting
stress on the bonds within a molecule, or by holding molecules close together. This
increases the likelihood of a reaction, and so lowers the energy required to begin it.
20
The activation energy of a reaction may be denoted by Ea. In relation to biology (such as
biochemistry), the activation energy (or energy of activation) pertains to the energy
needed to initiate a reaction. For instance, the activation energy required to breakdown
glucose into pyruvic acid in respiration is two ATP.
1.5.3
The ability of enzymes to lower the activation energy of a reaction is a result of their
structure. Enzymes are large proteins with complex, highly ordered, three-dimensional
shapes produced by physical and chemical interactions between their amino acid
subunits. Each type of enzyme has a characteristic three-dimensional shape, or
conformation, with ridges, grooves, and pockets lined with specific amino acids. The
particular pockets that are active in catalyzing a reaction are called the active sites of the
enzyme.
The reactant molecules, which are called the substrates of the enzyme, have specific
shapes that allow them to fit into the active sites. The enzyme can thus be thought of as a
lock into which only a specifically shaped keythe substratecan fit. This lock-andkey model of enzyme activity.
In some cases, the fit between an enzyme and its substrate may not be perfect at first. A
perfect fit may be induced, however, as the substrate gradually slips into the active site.
This model of enzyme activity, in which the enzyme undergoes a slight structural change
to better fit the substrate, is called the induced-fit model. The enzyme-substrate
complex, formed temporarily in the course of the reaction, then dissociates to yield
products and the free unaltered enzyme.
Since enzymes are very specific as to their substrates and activity, the concentration of a
specific enzyme in a sample of fluid can be measured relatively easily. This is usually
done by measuring the rate of conversion of the enzymes substrates into products
under specified conditions. The presence of an enzyme in a sample can thus be detected
21
by the job it does, and its concentration can be measured by how rapidly it performs its
job.
22
fits into it and an enzyme-substrate (ES) complex can form. The reaction is then
catalyzed and an enzyme-product (EP) complex forms.
1.6
1.6.1
23
Lets look at a few examples of this simple concept. Urea is the substrate acted on by the
enzyme urease.
Note that the name of this enzyme is simply the name of the substrate with the ending
ase added. With the exception of some historical common names, the general ending for
the name of an enzyme is ase. For instance lactose is the substrate of lactase. Thus
urease catalyzes hydrolysis of urea, lactase catalyzes hydrolysis of lactose to glucose and
galactose, and DNA polymerase catalyzes the polymerization of nucleotides to form
DNA.
Other enzymes may be named for the reactions they catalyze. For example,
dehydrogenases remove hydrogen atoms, transferring them to a coenzyme, and
decarboxylases remove carboxyl group.
The prefix de- indicates that a functional group is being removed. Hydrogenases and
carboxylases, on the other hand, add hydrogen and carboxyl groups. Some enzyme
names include both the name of the substrate and of the reaction type. For example,
lactate dehydrogenase removes hydrogen atoms from lactate ions, and pyruvate
decarboxylase removes the carboxyl group from pyruvate.
The names of many enzymes specify both the substrate of the enzyme and the job
category of the enzyme. Lactate dehydrogenase, for example, removes hydrogens from
lactic acid. Enzymes that do exactly the same job (that catalyze the same reaction) in
different organs have the same name, since the name describes the activity of the
enzyme. Different organs, however, may make slightly different models of the enzyme
that differ in one or a few amino acids. These different models of the same enzyme are
called isoenzymes. The differences in structure do not affect the active sites (otherwise
the enzymes would not catalyze the same reaction), but they do alter the structure of the
enzymes at other locations, so that the different isoenzymatic forms can be separated by
standard biochemical procedures.
Note: The complete name for lactate dehydrogenase is lactate:NAD oxidoreductase. This systematic
name tells us the substrate, coenzyme, and type of reaction catalyzed.
26
Figure 1.12: The enzyme classes. Most enzymes catalyze the transfer of electrons,
atoms, or functional groups. They are therefore classified, given code numbers, and
assigned names according to the type of transfer reaction, the group donor, and the
group acceptor.
1. Oxidoreductases
Oxidoreductases are enzymes that catalyze oxidation-reduction (redox) reactions.
Lactate dehydrogenase is an oxidoreductase that removes hydrogen from a molecule of
lactate. Other subclasses of the oxidoreductases include oxidases and reductases.
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2. Transferases
Transferases are enzymes that catalyze the transfer of functional groups from one
molecule to another. For example, a transaminase catalyzes the transfer of an amino
functional group, and a kinase catalyzes the transfer of a phosphate group. Kinases play
a major role in energy-harvesting processes involving ATP. In the adrenal gland,
norepinephrine is converted to epinephrine by the enzyme phenylethanolamine-Nmethyltransferase (PNMT), a transmethylase.
3. Hydrolases
Hydrolases catalyze hydrolytic reactions, that is, the addition of a water molecule to a
bond resulting in bond breakage. These reactions are important in the digestive process.
For example, lipases catalyze the hydrolysis of the ester bonds in triglycerides.
4. Lyases
Lyases catalyze the addition of a group to a double bond or the removal of a group to
form a double bond. Citrate lyase catalyzes the removal of an acetyl group from a
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molecule of citrate. The products of this reaction include oxaloacetate, acetyl CoA, ADP.
And inorganic phosphate group (Pi).
5. Isomerases
Isomerases rearrange the functional group within a molecule and catalyze the
conversion of one isomer into another. For example, phosphoglyceromutase converts
one structural isomer, 3-phosphoglycerate, into 2-phosphoglycerate.
6. Ligases
Ligases are enzymes that catalyze the condensation or joining of two molecules. For
example, DNA ligase catalyzes the joining of the hydroxyl group of a nucleotide in a
DNA strand with the phosphate group of the adjacent nucleotide to form a phosphoester
bond.
Solution: The reaction occurring here involves breaking a bond, in this case a peptide
bond, by the addition of a water molecule. The enzyme is classified as a hydrolase,
specifically a peptidase.
Solution: This is the first reaction in the biochemical pathway called glycolysis. A
phosphoryl group is transferred from a donor molecule, adenosine triphosphate, to the
recipient molecule, glucose. The products are glucose-6-phosphate and adenosine
diphosphate. The enzyme, called hexokinase, is an example of a transfers.
30
Solution: In this reaction, the reactant malate is oxidized and the coenzyme NAD+ is
reduced. The enzyme that catalyzes this reaction, malate dehydrogenase, is an
oxidoreductase.
Solution: Careful inspection of the structure of the reactant and the product reveals
that they each have the same number of carbon, hydrogen, oxygen, and phosphorus
atoms; thus, they must be constitutional isomers. The enzyme must be an isomerase. Its
name is triose phosphate isomerase.
References
1.
David L Nelson and Michael M Cox. 2008. Lehninger Principles of Biochemistry, Fifth Edition. WH
Freeman and Company, New York.
2. Victor Rodwell, David Bender, Kathleen M Botham, Peter J Kennelly, P Anthony Weil. 2015. Harpers
Illustrated Biochemistry, 30th Edition. The McGraw-Hill Companies, New York.
3. Eric P Widmaier, Hershel Raff and Kevin T Strang. 2010. Vander's Human Physiology: The
Mechanisms of Body Function, Twelfth Edition. The McGrawHill Companies, New York.
4. Stuart Ira Fox. 2014. Human Physiology. Thirteenth Edition. The McGraw-Hill Companies, New
York.
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