Task 3 (a and b)

In biochemistry, a metabolic pathway is a linked series of chemical reactions occurring within a cell.
The reactants, products, and intermediates of an enzymatic reaction are known as metabolites,
which are modified by a sequence of chemical reactions catalyzed by enzymes.[1]:26 In most cases of
a metabolic pathway, the product of one enzyme acts as the substrate for the next. However, side
products are considered waste and removed from the cell.[2] These enzymes often require dietary
minerals, vitamins, and other cofactors to function.
There are two types of metabolic pathways that are characterized by their ability to either synthesize
molecules with the utilization of energy (anabolic pathway) or break down of complex molecules by
releasing energy in the process (catabolic pathway).[6] The two pathways complement each other in
that the energy released from one is used up by the other. The degradative process of a catabolic
pathway provides the energy required to conduct a biosynthesis of an anabolic pathway.[6] In addition
to the two distinct metabolic pathways is the amphibolic pathway, which can be either catabolic or
anabolic based on the need for or the availability of energy.[7]
Pathways are required for the maintenance of homeostasis within an organism and the flux of
metabolites through a pathway is regulated depending on the needs of the cell and the availability of
the substrate. The end product of a pathway may be used immediately, initiate another metabolic
pathway or be stored for later use. The metabolism of a cell consists of an elaborate network of
interconnected pathways that enable the synthesis and breakdown of molecules (anabolism and
catabolism).

Energy is required for the normal functioning of the organs in the body. Many tissues can also
use fat or protein as an energy source but others, such as the brain and red blood cells, can
only use glucose.

Glucose is stored in the body as glycogen. The liver is an important storage site for glycogen. Glycogen is mobilized
and converted to glucose by gluconeogenesis when the blood glucose concentration is low. Glucose may also be
produced from non-carbohydrate precursors, such as pyruvate, amino acids and glycerol, by gluconeogenesis. It is
gluconeogenesis that maintains blood glucose concentrations, for example during starvation and intense exercise.

The first metabolic pathway that we encounter is glycolysis, an ancient pathway employed by a
host of organisms. Glycolysis is the sequence of reactions that metabolizes one molecule of
glucose to two molecules of pyruvate with the concomitant net production of two molecules
of ATP. This process is anaerobic (i.e., it does not require O2) inasmuch as it evolved before the
accumulation of substantial amounts of oxygen in the atmosphere. Pyruvate can be further
processed anaerobically (fermented) to lactate (lactic acid fermentation) or ethanol (alcoholic
fermentation). Under aerobic conditions, pyruvate can be completely oxidized to CO2, generating
much more ATP, as will be discussed in Chapters 17 and 18.
Glucose can be synthesized from noncarbohydrate precursors, such as pyruvate and lactic acid,
in the process of gluconeogenesis. Although glycolysis and gluconeogenesis have some of the
same enzymes in common, the two pathways are not simply the reverse of each other. In
particular, the highly exergonic, irreversible steps of glycolysis are bypassed in gluconeogenesis.
Both pathways are stringently controlled by intercellular and intracellular signals, and they are
reciprocally regulated so that glycolysis and gluconeogenesis do not take place simultaneously in
the same cell to a significant extent.
Our understanding of glucose metabolism, especially glycolysis, has a rich history. Indeed, the
development of biochemistry and the delineation of glycolysis went hand in hand. A key
discovery was made by Hans Buchner and Eduard Buchner in 1897, quite by accident. The
Buchners were interested in manufacturing cell-free extracts of yeast for possible therapeutic
use. These extracts had to be preserved without the use of antiseptics such as phenol, and so they
decided to try sucrose, a commonly used preservative in kitchen chemistry.
Metabolism (/məˈtæbəlɪzəm/, from Greek: μεταβολή metabolē, "change") is the set of life-
sustaining chemical reactions in organisms. The three main purposes of metabolism are: the
conversion of food to energy to run cellular processes; the conversion of food/fuel to building blocks
for proteins, lipids, nucleic acids, and some carbohydrates; and the elimination of nitrogenous
wastes. These enzyme-catalyzed reactions allow organisms to grow and reproduce, maintain their
structures, and respond to their environments. (The word metabolism can also refer to the sum of all
chemical reactions that occur in living organisms, including digestion and the transport of substances
into and between different cells, in which case the above described set of reactions within the cells is
called intermediary metabolism or intermediate metabolism).
Task 3c
Gluconeogenesis is much like glycolysis only the process occurs in reverse. However, there are
exceptions. In glycolysis there are three highly exergonic steps (steps 1,3,10). These are also
regulatory steps which include the enzymes hexokinase, phosphofructokinase, and pyruvate
kinase. Biological reactions can occur in both the forward and reverse direction. If the reaction
occurs in the reverse direction the energy normally released in that reaction is now required. If
gluconeogenesis were to simply occur in reverse the reaction would require too much energy to
be profitable to that particular organism. In order to overcome this problem, nature has evolved
three other enzymes to replace the glycolysis enzymes hexokinase, phosphofructokinase, and
pyruvate kinase when going through the process of gluconeogenesis:
1. The first step in gluconeogenesis is the conversion of pyruvate to phosphoenolpyruvic acid
(PEP). In order to convert pyruvate to PEP there are several steps and several enzymes
required. Pyruvate carboxylase, PEP carboxykinase and malate dehydrogenase are the
three enzymes responsible for this conversion. Pyruvate carboxylase is found on the
mitochondria and converts pyruvate into oxaloacetate. Because oxaloacetate cannot pass
through the mitochondria membranes it must be first converted into malate by malate
dehydrogenase. Malate can then cross the mitochondria membrane into the cytoplasm
where it is then converted back into oxaloacetate with another malate dehydrogenase.
Lastly, oxaloacetate is converted into PEP via PEP carboxykinase. The next several steps
are exactly the same as glycolysis only the process is in reverse.
2. The second step that differs from glycolysis is the conversion of fructose-1,6-bP to
fructose-6-P with the use of the enzyme fructose-1,6-phosphatase. The conversion of
fructose-6-P to glucose-6-P uses the same enzyme as glycolysis, phosphoglucoisomerase.
3. The last step that differs from glycolysis is the conversion of glucose-6-P to glucose with
the enzyme glucose-6-phosphatase. This enzyme is located in the endoplasmic reticulum.

Task 3d
Gluconeogenesis and glycolysis are coordinated so that within a cell one pathway is relatively
inactive while the other is highly active. If both sets of reactions were highly active at the same
time, the net result would be the hydrolysis of four nucleotide triphosphates (two ATP plus
two GTP) per reaction cycle. Both glycolysis and gluconeogenesis are highly exergonic under
cellular conditions, and so there is no thermodynamic barrier to such simultaneous activity.
However, the amounts and activities of the distinctive enzymes of each pathway are controlled
so that both pathways are not highly active at the same time. The rate of glycolysis is also
determined by the concentration of glucose, and the rate of gluconeogenesis by the
concentrations of lactate and other precursors of glucose.
Phosphofructokinase and fructose 1,6-bisphosphatase are also reciprocally controlled by fructose 2,6-
bisphosphate in the liver (Section 16.2.2). The level of F-2,6-BP is low during starvation and high in the
fed state, because of the antagonistic effects of glucagon and insulin on the production and degradation
of this signal molecule. During starvation, gluconeogenesis predominates because the level of F-2,6-BP is
very low. Glucose formed by the liver under these conditions is essential for the viability of brain and
muscle.

Task 4a

The first step in purifying intracellular (inside the cell) proteins is the preparation
of a crude extract. The extract will contain a complex mixture of all the proteins
from the cell cytoplasm, and some additional macromolecules, cofactors, and
nutrients. The crude extract may be used for some applications in biotechnology,
however, if purity is an issue, subsequent purification steps must be followed.
Crude protein extracts are prepared by removal of cellular debris generated by
cell lysis, which is achieved using chemicals and enzymes, sonication or a
French Press.

The debris is removed by centrifugation, and the supernatant is recovered. Crude

preparations of extracellular proteins may be obtained by simply removing the
cells by centrifugation.

In the past, a common second step to purifying a protein from a crude extract
was by precipitation in a solution with high osmotic strength (i.e. salt solutions).
Nucleic acids in the crude extract can be removed by precipitating aggregates
formed with streptomycin sulfate or protamine sulfate. Protein precipitation is
usually done using ammonium sulfate as the salt.

Different proteins will precipitate in different concentrations of ammonium

sulfate. In general, proteins of higher molecular weight precipitate in lower
concentrations of ammonium sulfate. Salt precipitation does not usually lead to a
highly purified protein but can assist in eliminating some unwanted proteins in a
mixture and concentrating the sample. Salts in the solution are then removed by
dialysis through porous cellulose tubing, filtration, or gel exclusion
chromatography.

Task 4b
 1) An iron-sulfur protein with properties similar to those of ferredoxins found in the leaves of
higher plants has been isolated from bean sprouts--a non-photosynthetic plant tissue.
The bean sprout protein has a molecular mass of 12.5 kDa and appears to contain a single
[2Fe-2S] cluster. The absorbance and circular dichroism spectra of
the bean sprout protein resemble those of spinach leaf ferredoxin and
the bean sprout protein can replace spinach ferredoxin as an electron donor for NADP+
reduction, nitrite reduction and thioredoxin reduction by spinach leaf enzymes. Although the
reduced bean sprout protein (Em = -440 mV) is a slightly stronger reductant than spinach
ferredoxin and appears to be less acidic than spinach ferredoxin, the two proteins are similar
enough so that the bean sprout protein is recognized by an antibody raised against spinach
ferredoxin.
2) Purification should yield a sample of protein containing only one type of molecule, the
protein in which the biochemist is interested. This protein sample may be only a fraction
of 1% of the starting material, whether that starting material consists of cells in culture or
a particular organ from a plant or animal. How is the biochemist able to isolate a
particular protein from a complex mixture of proteins?
The biochemist needs a test, called an assay, for some unique identifying property of the
protein so that he or she can tell when the protein is present. Determining an effective
assay is often difficult; but the more specific the assay, the more effective the
purification. For enzymes, which are protein catalysts (Chapter 8), the assay is usually
based on the reaction that the enzyme catalyzes in the cell. Consider the enzyme lactate
dehydrogenase, an important player in the anaerobic generation of energy from glucose
as well as in the synthesis of glucose from lactate.

Task 4c
There are several chemical tests available for the identification of the major types of organic
compounds in living organisms. Typically these tests are used to determine the makeup of an
unknown material. For instance, a forensic detective may be interested identifying a fluid found
at a crime scene. The collected fluid is the unknown. As the tests are carried out, the detective
will also use known compounds, or controls, for comparison. During the experiment the
detective compares the unknown's response to the experimental procedure with the control's
response to that same procedure.
Controls are important because they reveal the specificity of a particular test. For example, if
water and a glucose solution react similarly in a particular test, the test cannot distinguish water
from glucose. But if the glucose solution reacts differently from distilled water, the test can
distinguish water from glucose. In this instance, the distilled water is a negative control for the
test, and a known glucose solution is a positive control. A positive control contains the variable
for which you are testing. It produces a positive reaction and demonstrates the test's ability to
detect what you expect. A positive reaction to a positive control demonstrates that your test
reacts correctly. A negative control does not contain the variable for which you are searching. It
contains only the solvent (often distilled water with no solute) and does not react in the test. A
negative control demonstrates what a negative result looks like. There are literally hundreds of
tests available for biological molecules. In today’s lab we will be using some of the more
common methods to look for the presence of simple (or reducing sugars), starch, protein, lipid,
and DNA.

Task 4d
 maximum activity of protease PT121 was observed at 60°C, though considerable activity was also
observed in the temperature range 40 – 70°C (Fig. 4). The purified protease showed good stability
and retained more than 90% of its initial activity after incubation for 1 h from 30 to 50°C (Fig. 5).
However, the activity decreased to 75% after incubation for 1 h at 60°C. The protease activity rapidly
decreased after incubation at temperatures higher than 60°C (only 3% activity remained after
incubation at 70°C). The temperature optimum and thermostability of the protease were similar to
those of many other reported proteases from P. aeruginosa [24, 25]. The protease was further
characterized for its K m and V max towards casein as a substrate. The K m was 3.97 mg/ml, while
the V max was 7.58 μmol/min. Lower K m values on casein were found in those of P. aeruginosa
PseA protease (2.69 mg/ml) [23], P. fluorescens 22F protease (2.44 mg/ml) [26], Bacillus sp.
TKU004 protease (2.98 mg/ml) [27], and B. subtilis TKU007 protease (0.13 mg/ml) [22]. As regard to
V max of PT121 protease (7.58 μmol/min), which is far more than 0.14 of Bacillus sp. TKU004 [27],
0.86 of Bacillus subtilis TKU007 [22], 1.26 of Bacillus cereus [28], and 3.03 of P. aeruginosa PseA .