These lecture notes will provide an outline of information from the lectures. They are not complete. They should be used to help follow the lecture and as a guideline for information I think is important. You will need to fill in the gaps.


Chapter 5

I.    Enzymes A.    Are proteins, generally
1.    Some enzymes contain a nonprotein part, the COFACTOR, that is a metal ion (of magnesium, iron, or zinc)
2.    Some enzymes contain a nonprotein part, the COENZYME, that is an organic molecule (organic molecules contain carbon chains; examples include NAD, also known as nicotinamide adenine dinucleotide, and FAD, flavin adenine dinucleotide); these are important because they act as carriers of electrons
3.    The entire enzyme, whether it is just the protein molecule, or the enzyme and its cofactor or the enzyme and its coenzyme, is called the HOLOENZYME B.    Are catalysts
C.    Speed up chemical reactions
D.    Are specific
E.    Act on a substrate or substrates
F.    Catalyze only one reaction
1.    Reaction may be anabolic (synthesis of large molecules)
2.    Or, reaction may be catabolic (breakdown (digestion) of large molecules into smaller molecules)
 G.    Have an active site
1.    Specific region of the surface of the enzyme
2.    Substrate attaches here
3.    Result is a temporary intermediate compound, the enzyme-substrate complex
4.    The substrate is chemically changed here
a)    By rearrangement of existing atoms into a different product
b)    By its breakdown into smaller sized products
c)    By its combination with another substrate resulting in a more complex product
d)    This chemically changed substrate is released from the enzyme as a new end-product 5.    The unchanged enzyme is free to act on another substrate
6.    The clinical significance of an active site is that blocking the active site prevents the bacteria from performing that aspect of metabolism a)    Many active sites contain sulfhydryl groups (-SH)
b)    Disinfectants and antibiotics that react with sulfhydryl groups inactivate the enzyme by blocking its active site
c)    An example is sulfonamide drugs II.    Naming Enzymes A.    -ase ending is usually used
B.    Substrate acted on by enzyme may precede -ase ending (for example, lactase acts on lactose)
C.    Function performed by enzyme may precede -ase ending (for example, hydrolases are important in hydrolysis reactions) III.    Factors That Influence Enzymatic Activity A.    The clinical significance of these factors is that knowing what it takes to disrupt bacterial enzymatic activity can be used to make potent antibiotics or disinfectants
B.    Temperature
1.    The higher the temperature, the higher the rate of the reaction
2.    UNTIL DENATURATION OCCURS C.    pH
1.    Optimum pH
2.    Denaturation occurs with extremes in pH D.    Substrate Concentration
1.    The higher the substrate concentration, the higher the rate of the reaction
2.    UNTIL SATURATION OCCURS
a)    High substrate concentration saturates enzyme active sites
b)    When saturation has occurred, increasing substrate concentration will not increase rate E.    Inhibitors
 IV.   Energy and ATP A.    Energy is necessary to metabolism and bacterial life functions such as binary fission, flagellar motion, and spore formation
B.    In the bacterial cell (and, in ALL living cells), energy is deployed from a high energy molecule called adenosine triphosphate (ATP) 1.    ATP's energy is released when a specific enzyme causes one of its bonds to break (the bond that holds the third phosphate group onto the molecule)
2.    Breaking this bond causes the formation of adenosine diphosphate (ADP), a separate phosphate group, and a great deal of energy, which can be used to do the work of the organism a)    One mole of ATP (507 grams or about one pound) releases 7300 calories 3.    Where does the energy in the ATP come from? a)    Ultimately, the sun
b)    Those organisms that are capable of photosynthesis store the sun's energy in organic molecules such as carbohydrates
c)    Those organisms that are not capable of photosynthesis must consume organic molecules such as carbohydrates (made by photosynthetic organisms) and then break these molecules down in order to release the sun's energy (1)    THAT energy is then plugged into ATP V.    The Catabolism of Glucose (breakdown or digestion) A.    Takes place in all living things
B.    Involves a metabolic pathway 1.    A metabolic pathway is a series of chemical reactions, each of which is catalyzed by a specific enzyme
2.    Usually the end-product of one reaction serves as the substrate for the next reaction
3.    This is a "Rube Goldberg apparatus" kind of event (kind of a domino effect) C.    The catabolism of glucose also goes by another name, cellular respiration 1.    When cellular respiration takes place in the presence of oxygen, it is called AEROBIC cellular respiration
2.    When cellular respiration takes place in the absence of oxygen, it is called ANAEROBIC cellular respiration D.    The chemical equation of glucose catabolism under aerobic conditions (aerobic cellular respiration ) is represented by this equation: C6H12O6 + 6 O2 +38 ADP +38 P = 6 CO2 +6 H20 +38 ATP 1.    To get from the substrate, C6H12O6, to the end-product requires three major processes a)    Glycolysis
b)    Krebs cycle
c)    Oxidative phosphorylation 2.    Glycolysis a)    This is the chemical breakdown (digestion or hydrolysis) of glucose
b)    Glycolysis occurs in the cytoplasm of bacteria
c)    The substrate is glucose
d)    The end-products are 2 molecules of pyruvic acid and four molecules of ATP (1)    It took two molecules of ATP to get this reaction going, so we actually NET two molecules of ATP
(2)    Two of the co-enzymes, NAD+, are reduced to NADH+, which means that each NADH+ is now carrying energy from the glucose molecule in the form of 2 electrons and 2 protons e)    It takes nine steps and nine enzymes to get from glucose to pyruvic acid
f)    Although we are talking about aerobic respiration, oxygen is not required for glycolysis to take place (1)    In fact, glycolysis will occur in both aerobic and anaerobic cellular respiration in exactly the same way 3.    Krebs Cycle a)    Whereas glycolysis occurred in the bacterial cytoplasm, the Krebs Cycle takes place along the plasma membrane of the bacteria (in eukaryotes, this cycle takes place in the mitochondria)
b)    In the Krebs Cycle, the substrates are two molecules of pyruvic acid
c)    The end-products are six molecules of CO2 and two molecules of ATP (1)    If you have been keeping track, we netted two ATP during glycolysis and now we have another 2 ATP from the Krebs Cycle
(2)    In addition, more high energy electrons and protons are stored in six molecules of NADH+ and two molecules of FADH2 (another coenzyme electron carrier) (a)    Don't forget the two molecules of high energy NADH+ from glycolysis! 4.    Oxidative phosphorylation (also known as electron transport chain or chemiosmosis) a)    This takes place at the bacterial cell membrane (and in the mitochondria of eukaryotic cells)
b)    This is where MOST (34 of the 38) of the ATP molecules are formed
c)    Remember the NADH+ and the FADH2? They bring their high energy electrons and protons to CYTOCHROMES that are found in the cell membrane (1)    Cytochromes are molecules that contain iron ions that are able to accept and release electrons
(2)    These cytochromes pass the electrons back and forth among themselves, releasing the electrons' energy with each pass, until the electrons reach the final acceptor, OXYGEN (a)    If oxygen is not present, this entire process grinds to a halt
(b)    Oxygen is CRITICAL to the success of oxidative phosphorylation (3)    This energy is used to propel the protons (that accompanied the electrons) across the cell membrane and then to REENTER the bacterium through a protein molecule that is lined with ATP synthetase (guess what this does)
(4)    Each time a set of protons reenters through the protein molecule, THREE ATP molecules are formed
(5)    The oxygen (above) combines with a pair of re-entered protons (H+), and 6 molecules of water are formed (H2O) for every one molecule of glucose E.    What if there is no oxygen? 1.    The organism must be capable of anaerobic cellular respiration to survive
2.    In anaerobic cellular respiration, the final electron acceptor is an inorganic molecule such as nitrate (NO3-), sulfate (SO4=), or CO2
3.    There is glycolysis, a Krebs Cycle, and oxidative phosphorylation a)    HOWEVER, because nitrate, sulfate, and CO2 are not as good at accepting electrons as oxygen is, the net yield of ATP molecules is less than it is with aerobic cellular respiration
b)    Take home message: anaerobic cellular respiration is not as efficient in its breakdown of glucose; because fewer ATP are generated, the organism has less energy to perform life's functions and is consequently slower growing than its aerobic counterpart F.    Fermentation 1.    This is NOT cellular respiration a)    Cellular respiration involves oxidative phosphorylation, which fermentation does not do
b)    It is an anaerobic process, because it takes place in the absence of oxygen 2.    Fermentation produces the least amount of ATP because the organism does not respire (go through oxidative phosphorylation)
3.    Fermentation is used by facultative anaerobes when they find themselves in an environment that does not contain a suitable inorganic final electron acceptor
4.    Fermentation is also used by those organisms that completely lack the necessary enzymes that would permit oxidative phosphorylation to occur
5.    In fermentation, glycolysis occurs as described above
6.    The two pyruvic molecules are then INCOMPLETELY oxidized to a final organic molecule such as lactic acid, ethanol, butyric acid, propionic acid, and acetic acid
7.    Fermenters have great commercial significance a)    Food manufacturers purposely encourage some microbes to flourish in certain food products in order to produce cheeses, sour cream, alcoholic beverages, and soy sauce 8.    This is the LEAST efficient method of generating energy-rich ATP; only two ATP are produced from glycolysis VI.    The Anabolism of Protein A.    Proteins are made up of amino acids joined together by peptide bonds 1.    The chain of amino acids formed when a large number of amino acids are joined by peptide bonds is called a polypeptide chain (most proteins contain about 400 amino acids in their polypeptide chain)
2.    The number and arrangement (sequence) of amino acids in a protein determines its PRIMARY structure
3.    Once the primary structure is in place, amino acids will arrange themselves in a two-dimensional fashion: some may twist themselves around; others take on a pleated, accordion-type of arrangement-the arrangement that a protein's amino acid take determines its SECONDARY structure
4.    After assuming its secondary structure, a protein will fold into and upon itself in a three-dimensional fashion-this is called its TERTIARY structure
5.    Finally, some proteins are so complex that they require more than one polypeptide chain (each with its own primary, secondary, and tertiary structure) to join together; the resulting shape produced by the combination of polypeptide chains is called the QUATERNARY structure of the protein (not all proteins have a quaternary structure) B.    There are 20 different amino acids; their sequence is determined by the master molecule, deoxyribonucleic acid (DNA) 1.   A Review of DNA structure a.    DNA is a macromolecule that makes up the chromosome found in bacteria
b.    Its basic building block is the nucleotide
1)    One nucleotide is composed of deoxyribose (sugar), a phosphate group, and a nitrogen base
2)    The nucleotide is named accourding to its nitrogen base a)   The four types of bases are adenine, thymine, cytosine, and guanine, abbreviated A, T, C, and G c.   Nucleotides are twisted together in pairs to form a double helix (spiral staircase) 1)    The sides of the spiral are alternating sugar and phosphate groups (sugar-phosphate backbone)
2)    The rungs of the ladder are the nitrogenous bases
3)    Each nitrogenous base is attached to a sugar
4)    The rungs are held together by hydrogen bonds between nitrogenous bases d.    Nitrogenous bases always pair up in a specific way
1)    A to T
2)    C to G
3)    Thus the base sequence of one DNA strand determines the sequence of the other strand e.    The two strands of DNA are complementary to each other 2.    Segments of DNA that contain the code for a particular protein (that is, its sequence of amino acids) are called "genes"
3.    In protein synthesis, each gene's code is first TRANSCRIBED by messenger ribonucleic acid (mRNA) a.   Recall that RNA differs from DNA in several ways
b.    Instead of deoxyribose for the sugar, there is ribose
c.    Instead of being double-stranded, RNA is single-stranded
d.    Instead of the nitrogen base thymine, RNA contains uracil
1)    Thus wherever adenine would pair with thymine in DNA, you will find adenine pairing with uracil in RNA e.    RNA comes in three forms, mRNA, rRNA, and tRNA
f.   Transcription is the synthesis of a complementary strand of mRNA from a specific gene on the DNA template 1)    Recall that a gene is the sequence of nitrogenous bases of DNA that contains genetic information for making a particular protein
2)    This is how genetic information is used within a cell to produce the proteins the cell needs to function g.    Transcription begins with the uncoupling of the double-stranded DNA molecule 1)   The uncoupled DNA molecule's nitrogen bases are now exposed and serve as the template for the mRNA h.   Transcription starts with RNA polymerase binding to the DNA at a site called the promoter 1)    The promoter is found on only one of the two un-wound DNA strands i.   The language of mRNA is in the form of codons 1)    A codon is a group of three nucleotides
2)    Thus, AUG, GGC, and AAA are all examples of codons
3)    It is the sequence of codons on the mRNA molecule that will ultimately determine the sequence of amino acids that will be present in the protein that is to be synthesized j.    Each codon codes for a particular amino acid 1)    This is the infamous genetic code
2)    The genetic code is considered to be degenerate because although there are 64 possible codons (4x4x4 possible ways to combine), there are only 20 amino acids
3)    This means that most amino acids are signaled by several alternative codons
4)    Degeneracy allows for a certain amount of change (mutation) in the DNA without affecting the protein product k.    There are 3 nonsense codons 1)    Nonsense codons do not code for amino acids, but are used as punctuation, signaling the end of the sequence of nucleotides for a particular protein molecule l.    61 sense codons code for amino acids 1)    One of them, methionine, also substitutes as a "start" codon
2)    Methionine is often removed later, so not all proteins will begin with it 4.    Transcription uses complementary base pairing as the guide and the enzyme RNA polymerase to assemble free nucleotides into a new chain a.    The RNA nucleotides are positioned in a complementary fashion to the DNA exposed nitrogen bases, forming a chain of RNA that is complementary to the DNA segment that is being transcribed
b.    Each triplet of DNA nitrogen bases is transcribed to a corresponding codon on the mRNA
c.    RNA polymerase moves along the DNA, as the new RNA chain grows, until it arrives at the site on the DNA called the terminator
d.    Once at the terminator, transcription stops, and the RNA polymerase and the newly formed, single-stranded mRNA are released from the DNA and move to the ribosome where TRANSLATION will occur (1)    Transcription example: if gene on DNA has a nitrogen base sequence of TACGGGCATGTA, then the transcribed mRNA's nitrogen base sequence will be AUGCCCGUACAU (notice that RNA differs from DNA in that it has no thymine; instead, uracil is found wherever thymine would be found in DNA) 5.    Translation takes place at the ribosome, where mRNA directs the assembly of amino acids into a polypeptide chain a.    This is the actual assembly of amino acids into a protein molecule, using the mRNA as the source of information for the amino acid sequence
b.    At the ribosome, mRNA meets tRNA molecules , each of which is bound to a particular amino acid
c.    As the ribosome moves along the mRNA, each codon is exposed and matched to a complementary anticodon on the tRNA
d.    Thus, the amino acids, each of which is attached to a tRNA, are lined up in a sequence determined by the mRNA codons
e.    Recall that the sequence of mRNA codons was originally determined by the sequence of DNA triplets
f.    The amino acids are joined to each other by a peptide bond
g.    When the mRNA is fully translated, the polypeptide breaks away and twists into its secondary structure and then folds into its tertiary structure
h.    Translation example: DNA:
TACGGGCATGTA transcribed to
AUGCCCGUACAU, which is translated by a tRNA
UACGGGCAUGUA, that is attached to amino acids
methionine proline valine histidine (see code on page 153) VII.    The Control of Protein Synthesis A.    Some proteins are essential and made all the time 1.    These are called constitutive and the DNA genes that encode them are always active B.    Other proteins are not made all the time 1.    Some are inducible a.    The enzyme is made only when its substrate is present in the environment 2.    Others are ordinarily synthesized but can be repressed a.    When the end-product for a particular reaction is present to an excess in the environment, it causes the transcription of the enzyme that acts upon the substrate that produces the product to cease C.    One entire gene is divided into three parts 1.    Promoter a.    The region of DNA where RNA polymerase initiates transcription
2.    Operator a.    Acts as a 'go' or 'stop' signal for transcription of the structural gene(s) that follow it 3.    Structural gene or genes a.    This is the part that actually codes for the amino acids 4.    Together, these three parts constitute the OPERON
5.    The control region consists of the promoter and operator
6.    Repressor genes are further away from the structural gene and they control the operator gene a.    Repressor genes code for one of two types of repressor proteins (1)   One type of repressor protein blocks transcription from occurring by attaching to DNA at the operator region, just in front of the promoter a)    This prevents RNA polymerase from progressing past that region
b)    Transcription is initiated only if the repressor protein is not attached (2)   The other type of repressor protein blocks transcription from occurring only after a COREPRESSOR attaches to it (a)    This prevents RNA polymerase from progressing past that region in a similar fashion
(b)    When there is no corepressor present, RNA polymerase can initiate transcription b.    In the first example, anytime a particular substrate is not present in the environment, there is no reason to waste energy on building the enzyme that acts upon the substrate (1)    The repressor gene is transcribed by the mRNA, forming a repressor protein
(2)    This repressor protein attaches to the operator gene and overlaps the promoter region
(3)    As a result of this attachment, RNA polymerase cannot stimulate the transcription of the structural gene and the enzyme's code is not transcribed; translation does not occur; amino acids are not joined together; no protein synthesis occurs
(4)    Now, let's say the environment changes, and the substrate (for example, lactose, a sugar) is now present
(5)    The substrate (lactose) will bind to the repressor protein, inactivating it (a)    Substrates that can do this are called inducers
(b)    The enzyme that can now be transcribed is called inducible, because it was induced to be transcribed (6)    Now the inactivated repressor protein cannot attach to the operator/promoter area
(7)    The operator and promoter areas now stimulate the structural gene to allow transcription to occur; mRNA is formed; it is translated at the ribosome; the appropriate sequence of amino acids is joined by peptide bonds; the protein is fully synthesized and can work its magic upon the substrate c.    Conversely, in the second example above, an end-product (such as tryptophan) may be present in excess in the environment, and the organism no longer requires the enzyme that acts on the substrate that produces this end-product (1)    In this instance, the end-product (tryptophan) binds to the repressor protein itself
(2)    This binding causes the repressor protein to be activated, and it now attaches to the operator region
(3)    As a result of this attachment, RNA polymerase cannot stimulate the transcription of the structural gene and the enzyme's code is not transcribed; translation does not occur; amino acids are not joined together; no protein synthesis occurs D.    Summary 1.    Some proteins are made all the time
2.    Some proteins must be induced to form (their genes are turned off until inducers inactivate the off switch)
3.    Some proteins are repressed from forming (their genes are turned on until corepressors attach to the repressor proteins and activate the off switch)






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