Biochemistry Essay

The essence of nucleotides in cellular metabolism is signified by the fact that nearly all cells can synthesize them anew (de novo) and from the remnants of hydrolyzed nucleic acids. In this section of the essay, I will consider certain questions pertaining to nucleotide metabolism.      

1. Nucleotide metabolism.
What is the salvage pathway for synthesis of purine nucleotides
The biosynthesis of nucleotides in most cells occurs via the nearly ubiquitous de novo synthetic pathways. In this pathway, nucleotide biosynthesis starts from amino acids and their derivatives. However, a number of cells possess the ability to synthesize nucleotides from degraded nucleosides and nucleobases (adenine, guanine, and hypoxanthine).  Because these processes involve the reconversion of free purine and pyrimidine rings to their corresponding nucleotides, the process is called the salvage pathways.

The de novo synthesis of purine nucleotides and pyrimidine nucleotides is highly regulated, with multiple levels of feedback and feed forward regulation.  Define and give examples of these types of regulation as pertaining to nucleotide biosynthesis.

The most important type of feedback regulation is feedback inhibition (retroinhibition inhibition). It involves the inhibition of an enzyme by the accumulation of a product produced along the pathway of which the enzyme is part. In the de novo synthesis of purine nucleotides and pyrimidine nucleotides, feedback inhibition is seen in the first committed step in purine biosynthesis.  The step is involves the conversion of PRPP into phosphoribosylamine by glutamine phosphoribosyl amidotransferase. This essential step in feedback-inhibited by many purine ribonucleotides. It should be noted that AMP and GMP, the final products of the pathway, act synergistically in inhibiting the amidotransferase.

In addition, in the biosynthesis of AMP and GMP, Inosinate is the branch point. The biochemical reactions leading away from inosinate are sites of feedback inhibition. AMP inhibits the conversion of inosinate into adenylosuccinate, its immediate precursor. Similarly, GMP inhibits the conversion of inosinate into xanthylate, its immediate precursor.

On the other hand, feed forward regulation is any feuture of a metabolic pathway whereby information about the input to the system is used to influence the output of the system. In 1st step in nucleotides biosynthesis, ribose-5-phosphate transformed into IMP, a precursor for GTP and ATP, the enzyme amidophosphoryl transferase is stimulated allosterically by PRPP (its substrate) in a process called feed forward activation. Thus the reciprocal substrate relation which tends to regulatebalance the biosynthesis of adenine and guanine ribonucleotides is an example of feed forward regulation.

What is the biochemical logic of such elaborate regulation of synthesis of the purine and pyrimidine nucleotides

The logic of this complex regulation is to supply a critical balance of the four deoxyribonucleotides needed by the cell for the synthesis of DNA (pp.339).

2.  DNA Structure and Replication.
Biochemical composition of DNA and its 1o, 2o and 3o structure.  
Deoxyribonucleic acids have three major components, a sugar, a phosphate and a base. Typically, DNA macromolecules are linear polymers that consist of 4 bases two bases are derivatives of purine guanine (G) and adenine (A) and two are derivatives of pyrimidines  cytosine (C) and thymine (T).
Fig 1 showing structures of purines and pyrimidines.    
In the linear polymer, the nucleotides are linked by phosphodiester bridges  i.e. esterification of the 3-OH group of the sugar moiety into a phosphate group provides a joining point to the 5-OH group of adjacent sugar moiety. This process forms a chain of sugar linked by the phosphodiester bridges called a backbone.

In the Watson and crick structure, the essential features are two polynucleotide chains run in an antiparallel direction and coil around a common axis to form right handed double helix.  In this structure, the bases are on the inside while the sugar and the phosphate group are on the outside. Further, Thymine (T) is paired with Adenine (A), and cytosine (C) is paired with Guanine (G). The Adenine  cytosine base pair is held together by a pair of hydrogen bonds, and the Guanine Cytosine base pair by three hydrogen bonds (pp.854). See below.

Fig 2 showing base pairing in DNA (Adopted from Stryer 2004)
Further, the adjacent bases which are nearly perpendicular to the helix axis are separated by 3.4 . The helical structure repeats every 34 , thus we have 10 bases per helical turn, and a helical diameter of 20 . The sequence of DNA in this polynucleotide determined the nature of information stored. In addition, it must be noted that other conformations are possible.
DNA replication.

In a chemical sense, DNA replication is the process by which an exact copy of a template DNA (DNA molecule having a specific base sequence) is synthesized. The basic mechanism of replication in eukaryotes and prokaryotes has been elucidated at the molecular level. In general, DNA replication is more complex in eukaryotes than in prokaryotes. In this paper I will confine myself to replication of circular DNA in prokaryotes.

To summarize, the process of DNA replication is as follows DNA replication is semi-conservative and takes place in a replication fork (a Y-shaped structure). A DNA polymerase with proof reading and self correcting capabilities catalyzes nucleotide polymerization in a 5 to 3 prime directions. Given the fact that the complementary strands of DNA are antiparallel, the 5 to 3 prime DNA synthesis can take place continuously in the leading strand and discontinuously in the lagging strand. Because DNA polymerase require a pre-existing OH in the 5 , The DNA fragment in the lagging strand are primed by a short RNA primer molecules that are subsequently erased and replaced with DNA.

Further, DNA replication requires the cooperative activity of a number of proteins. The proteins include (1) DNA polymerase and DNA primase to catalyze nucleoside triphosphate polymerization (2) DNA helicases and single-strand DNA-binding (SSB) proteins to help in unwinding the double helix so that it can be replicated (3) DNA ligase and an RNAse (for degrading RNA primers) to stitch together the Okazaki fragments and (4) DNA topoisomerases to ease tensions and tangling in the DNA molecules associated with helical unwinding (pp 1021-1030).

A more elaborate description of the process of DNA replication in prokaryotes is as follows
(i) Initiation.

Initiation begins the unwinding of double helix DNA at sites called origin of replication. Several proteins including the initiator proteins and DNA helicase act in concert to unwind the double helix. This unwinding creates a replication bubble which has two replication forks (see fig below). The single strands in the bubble can now serve as templates.  
Actual replication is achieved by the DNA polymerase which adds nucleotides, successively, on the growing end of the strand. The enzyme operates according to three strict rules. First in can only copy single stranded DNA, second, it can only add DNA to the end of an existing chain. Third, it functions only in the 5 to 3 direction. The formation of the replication bubble satisfies the first requirement and a primase synthesizes an RNA primer that satisfies the requirement for an already existing chain.    

(ii) Elongation
This involves the linking together of complementary DNA (T-A, G-C base pairs) in a process determined by the sequence of the template strand. The main reaction is a phosphoryl group transfer with the 3-OH group acting as the nucleophile. Nucleophilic attack occurs at the alpha phosphorous of the incoming dNTP and inorganic pyrophosphate is released in the process. The general reaction is as follows

In this process, DNA polymerase catalyzes the joining of together of correctly paired nucleotides to the 3 OH end of the RNA primer and then continues to add successive nucleotide to the growing chain. (See fig below for illustration.)

Fig 3 showing DNA polymerase activity    (Adopted from Stryer 2004).

As DNA replication proceeds, Helicase successively unwinds the DNA. The DNA polymerase can then move progressively along the replication fork on the leading strand.
See fig below.

Fig 4. Replication Fork (Adopted from Stryer 2004)
Since the polarity of the DNA on the lagging strand is opposite that of the leading strand, the DNA must travel in the opposite direction to that of the leading strand. The answer to this problem is that DNA in the lagging strand is synthesized discontinuously as small fragments of about 1000 bases (Okazaki fragments). To allow DNA polymerase to act, the synthesis of each Okazaki fragment is initiated by the synthesis of a short primer RNA by the primase. DNA polymerase then attaches nucleotides to the new primer, thereby creating an Okazaki fragments that extends as far as the 5 prime end of primer. Finally, an RNAse degrades the RNA primer and a ligase joins successive strands. DNA replication is complete (pp. 1020-1030)

3. A portion of specific anti-sense strand of DNA molecule consists of the following sequence of nucleotide triplets.
In short, the given sequence is expressed as shown.
3 -TAC GAA CTT GGG TCC - 5   (anti-sense strand of DNA)
5- ATG CTT GAA CCC AGG  3 (Sense strand of DNA)


N-terminus - methionine - leucine - glutamic acid - proline  arginine  C-Terminus

DNA encoded information is expressed into mRNA by a transcriptional process. The process is catalyzed by an RNA polymerase and involved four key events promoter sequences located on the upstream side of the start site signal to the polymerase where to begin RNA polymerase add nucleotides to the mRNA in a 5 to 3 direction finally, terminators stop the transcription by the RNA polymerase.

For transcription to proceed in the given strand, RNA polymerase must bind to the DNA fragment. Binding occurs at the promoter sites and several proteins mediate this process. . After binding, the RNA polymerase unwinds the double helix exposing the anti sense strands that will serve as the template. Initially, the RNA polymerase aligns the first two nucleotides at the 5 end of the mRNA. Unlike the DNA polymerase, they do not need primers and they do not posses proof reading activity. The newly synthesized transcript grows in a 5 to 3 direction. After aligning the first two ribonucleotides, the RNA polymerase catalyses the formation of a phosphodiester bond between the two nucleotides, forming a diribonucleotide. Soon after, - subunit dissociates from the holoenzyme signaling the end of initiation.

After the formation of the first phosphodiester bond between A and U, elongation of the mRNA begins. The RNA polymerase extends the mRNA in the 5  to 3  direction, the 3  OH group of the RNA   DNA duplex is oriented such that it can attack the a-phosphorus atom of an incoming dNTP. The region of the DNA unwound by the enzyme is called the transcription bubble. In this way the AUG CUU GAA CCC AGG sequence will be transcribed.

Termination of transcription is signaled by specific sequences. In this process, the mRNA dissociate from the DNA-RNA hybrids and the enzyme complex. Regardless, other proteins also mediate this process.

Translation takes place in the ribosomes that coordinate the movement of aminoacyl- tRNA with the information encoded in the mRNA. The process consists of three key phases Initiation, elongation and termination.

Typically, translation begins with the coming together of the 30S subunit, the Shine-Delgarno sequence and the mRNA. After the formation of the complex, the initiator tRNA which carries fmethionine binds to the initiator AUG codon, and the 50S ribosomal subunit associates with its cognate 30S subunit thereby forming 70S ribosome. To understand how the polypeptide chain increases in length, we have to understand the structure of the ribosomes. Here, three sites provide a clue. The ribosome contains a P site where the initiator tRNA binds. An activated tRNA with an anticodon complementary to the codon in the A site on the ribosome then binds. This sets the stage for peptide bond formation the fmethionine molecule conjugated to the initiator tRNA will be transferred to the NH2-group of the amino acid in the A site. This transfer occurs in a ribosomal site called the peptidyl transferase center. The NH2-group of the charged -tRNA in the A site is oriented in such a way that it can hydrolyze the ester linkage between the initiator tRNA and the fmethionine molecule thereby forming a peptide bond and free tRNA. The free energy necessary for this process comes from the ATP that is cleaved by the aminoacyl-tRNA synthetase before the arrival of the charged-tRNA at the ribosome (pp 963-1000).
With the formation of the peptide bonds, the nascent peptide chain is now conjugated to the tRNA in the 30S subunits A site while a change in the interaction with the 50S subunit has placed that tRNA and its peptide in the P site of the 50S subunit (pp. 995). Since tRNA positioned at the P site of the 30S ribosomal subunit is now uncharged, translation can only proceed once the mRNA is translocated in a way which brings the codon for the next amino acid (CUU) to be added is in the A site. This process is mediated by an enzyme called elongation factor G, in a reaction driven by the hydrolysis of GTP. On the completion of translocation, the peptidyl-tRNA is now positioned on the P site, and the initiator tRNA (now uncharged) is now positioned in the E site and is not linked to the mRNA. The dissociation of the initiator tRNA returns the ribosome to its initial state, the only difference is that the peptide chain is attached to a different tRNA (in the DNA fragment given this aminoacyl-tRNA is tRNAleu). In the entire process, the peptide chain remains in the P site on the 50S subunit throughout the cycle. The next step in the translation of the mRNA is basically a repetition of this cycle.

Indeed, the entire peptide (methionine- leucine - glutamic acid - proline  arginine) will be synthesized by a repetition of this process (pp. 997).
An illustration of the process is given in the fig shown below.

Fig 5. Translation mechanism The cycle begins a charged tRNA in the A site and a peptidyl-tRNA in the P site. After the occupation of the two sites a peptide bond is formed. The tRNAs and the mRNA are the translocated in a process mediated by the elongation factor G, which shifts the uncharged-tRNA to the E site. Once there, it can dissociate and complete the cycle.                                     (Adopted from Stryer 2004)

4.   Traditionally RNA molecules have been subdivided into three groups ribosomal RNA (rRNA), messenger RNA (mRNA) and transfer RNA (tRNA). In recent years a number of other types of RNA have been discovered and are assuming greater importance in our understanding of living systems. The following are some of the examples.

(i) small nuclear RNAs (snRNAs)
snRNA function in a variety of cellular nuclear processes, including the splicing of pre-mRNA. Currently, five U-rich snRNAs, designated U1, U2, U4, U5, and U6, are known to participate in pre-mRNA splicing. Lengthwise, snRNAs range in length from 107 to 210 nucleotides. These RNA molecules are associated with 6 to 10 intra-nuclear proteins thereby forming small nuclear ribonucleoprotein particles (snRNPs)  remember that this applies only to eukaryotic cells. Incontrovertible evidence for the function of U1 snRNA in splicing was demonstrated in experiments which showed that that base pairing between the 5 splice site of a pre-mRNA and the 5 region of U1 snRNA is required for RNA splicing (Valadkhan S. 2010).

(ii) microRNAs (miRNAs)
In humans microRNAs appear to regulate about a third of human protein coding genes. Once synthesized by RNA polymerase II and polyadenylated and capped. The precursor miRNA are processed after which the mature miRNA are complexed with a set of proteins to form RNA-induced silencing complex (RICP). The RICP once formed, searches and base pairs with complementary nucleotide sequences in target mRNA. This process is greatly facilitated by Argonaute proteins, a component of RISC, which optimally positions a 5 region of miRNA thereby facilitating base pairing to target mRNA. One miRNA is bound to mRNA several things can result extensive base pairing of the miRNA to the mRNA leads to the cleaving of the mRNA by the Argonaute protein, this process removes the poly-A tail of the mRNA and exposes it to exonucleases. Following cleavage of the mRNA the RISC complex can seek out other mRNA. On the other hand, if the base pairing between miRNA and mRNA is less extensive, RNA translation is repressed and mRNA is destabilized (John G. et al 2003).

(iii) small interfering RNAs (siRNAs).
In general, siRNAs switch off gene expression by triggering the degradation of specific mRNAs and the establishment of compact chromatin structures. As to the discovery of siRNAs, they were discovered quite unexpectedly during attempt by researchers to manipulate, experimentally that is, the expression of specific genes.  

Researchers attempted to inhibit the expression of a specific C. elegans gene by microinjecting a single-stranded, complementary RNA (ssRNA) that base pair with transcribed mRNA and inhibit its translation, in the control set up, perfectly base-paired double-stranded RNA (dsRNA) was found to be more effective in inhibiting gene expression than the single stranded antisense oligonucleotides. Soon it was discovered that introduction of double stranded RNA can inhibit gene expression in plants. In the two cases, the dsRNA stimulated the degradation of all cellular RNAs containing complementary sequences. Subsequent experiments in Drosophila embryos demonstrated that long dsRNA that are involved in interference are initially processed into double stranded RNA intermediate loosely referred to as short interfering RNA (siRNAs). SiRNAs strands contain 2123 nucleotides that are hybridized to each other in such a way that the bases at the 3 end of either strand are single-stranded. The discovery that Dicer ribonuclease is necessary for the formation of siRNAs suggested that there is a relationship between RNA interference and miRNA-mediated translational repression. Recent biochemical studies have demonstrated that double-stranded siRNAs and miRNAs are part of a RNA-induced silencing complex (RISC). This multiprotein complex hydrolyses target RNAs that are complementary to their corresponding single-stranded siRNAs. Thus it acts in a similar mechanism to miRNA (John G. et al 2003).

5. Compartmentalization of eukaryotic cells leads to metabolic efficiency, but it also creates the potential for metabolic alterations due to defects in transport between compartments.  From what you have learned, predict the effects on metabolism and on the organism of the following defects

Export of glucose from the liver
To answer this question, I will limit myself to what happens in individuals with a glycogen storage disease called Von Gierkes Disease (Type I Glucose-6-phosphatase deficiency). The deficiency of Glucose-6-Phosphatase results in an increased level on intracellular Glucose-6-Phosphate (G6P). This, in turn, leads to a large accumulation of glycogen in liver and kidney (remember that G6P activates glycogen synthase and inhibits glycogen phosphorylase) and the inability to increase plasma glucose concentration in response to glucagon and epinephrine. The symptom of this inherited disease include massive liver enlargement and severe hypoglycemia after a limited period of fasting. Due to hypoglycemia, hormones, such as glucocorticoids are secreted which trigger an excessive mobilization of muscle amino acids which enter the liver. Secondly it triggers the activity of hormone sensitive lipases which result in the release of fatty acids from adipose tissue leading to the formation of ketone bodies.  Further, and because of hypoglycemia, citric acid cycle activity is reduced thereby blocking all the normal roots for the disposal of pyruvate. This forces the system towards lactate production leading to lactic acidosis.    

Transport of fatty acids into the mitochondria
A disease which typifies this state is carnitine deficiency that is caused by absence of carnitine transporter. The deficiency of intracellular carnitine impairs the translocation of long-chain fatty acids into the mitochondrial matrix. As a consequence, fatty acids are not available for -oxidation and ATP generation. And thus, leads to the intramitochondrial accumulation of acyl-CoA esters. This in turn, affects other intermediary metabolic pathways that require Acetyl-CoA (e.g. citric acid pathway, amino acid metabolism, pyruvate).

Low Density Lipoproteins (LDL) receptors
Dysfunctional (read deficient) LDL receptors result in excessive level of plasma cholesterol and ultimately leads to Atherosclerosis -atherosclerosis is a progressive disease that starts off as intracellular lipid deposits in the smooth muscle cells of inner arterial wall resulting in myocardial infarction. These deposits eventually become fibrous, calcified plaque that narrow and even block the artery (Voet et al 2004, pp 324). The strong correlation between the development of atherosclerosis and the level of plasma cholesterol is well exemplified in individuals with familial hypercholesterolemia (FH). Homozygotes for this inherited disorder have excessively high levels of cholesterol rich LDL in their plasma. The high level of Plasma LDL is traceable to two related causes. (1) The decreased rate of LDL degradation due to lack of LDL receptors. (2) The increased rate of LDL synthesis from IDL due to failure of LDL receptors to mop up IDL. The situation results in the formation of xanthomas (deposition of cholesterol in skin and tendons) and atheromas.            

6. What is signal transduction and how do the terms cascade and amplification apply to this field.
To understand the meaning of signaling transduction, we ought to remember that each signaling pathway has a starting point and an endpoint. The so-called start points are normally plasma membrane associated proteins (receptors) that function as receivers and sensors and of extra cellular signals. The receptors not only detect the signals but also convert them into signals that can be recognized and processed further within the intracellular environment (i.e. by generating another messenger to propagate the signal through the intracellular environment). This conversion process is called signal transduction.

On the other hand, a cascade is a sequence of successive activation reactions involving enzymes, hormones or both. The step by step reaction involving the formation of and mediation of the action of 2nd messengers, e.g. in the activation of glycogen phosphorylase is a good example.

Further, Amplification involves the increase in the strength of a chemical signal. To illustrate this concept, consider this one hormone molecule binds to a single receptor and initiates the activation of ten transducer proteins. Each transducer molecule activates ten enzymes molecules and each active enzyme produces ten active second messengers.      

7. Write out a detailed pathway of the hormonal control of triacyglycerols catabolism in adipose through the oxidation of released fatty acids.  Start with the binding of a necessary hormone to its receptor and ending with acetyl-CoA product. Remember to include allosteric effectors for your pathway.

Fatty acid oxidation (-oxidation) is largely regulated by the plasma concentration of fatty acids, which, in turn, is regulated by the hydrolysis rate of triacyglycerols in adipose by hormone-sensitive triacyglycerols lipase (Voet 2004. pp 690). Epinephrine, Nor-epinephrine and glucagon act to increase the concentration of cAMP in adipose tissue. cAMP allosterically activates cAMP-dependent protein kinase (cAPK), which in turn phosphorylates enzymes such as hormone sensitive lipases. Activated hormone sensitive lipases stimulate lipolysis in adipose tissue (pp 690) increasing plasma concentration of fatty acids and ultimately activating the beta-oxidation pathway.

See the figure below for an illustration of how the triacyglycerols are degraded to acetyl-Co in the -oxidation pathway.

Fig 5. Fatty acid oxidation (adopted from Stryer et al 2004)
AMP-dependent protein kinase (AMPK) acting in concert with cAMP dependent kinase also inhibits the activity of acetyl-CoA carboxylase by triggering its phosphorylation (acetyl-CoA catalyses the fast committed step rate controlling step in fatty acid anabolism). In this way, cAMP dependent phosphorylation simultaneously triggers -oxidation and inhibits fatty acid synthesis.

In contrast, insulin has an opposite effect to glucagon and epinephrine. For instance, it decreases the concentration of cAMP levels. This leads to the inactivation of hormone-sensitive lipases, thereby lowering the concentration of fatty acid available for -oxidation, it also inactivates acetyl-CoA.  The Glucagon-insulin ratio is therefore critical in determining the rate and direction of -oxidation.

Another regulatory point that inhibits -oxidation when fatty acid synthesis is triggered is the inhibition of carnitine palmitoyl transferase by malonyl-CoA. This inhibition limits the transport of the fatty acids into the mitochondrion and thus prevents    -oxidation (pp 690).

8.  What are the differences in metabolism between a patient with normal glucose metabolism and an uncontrolled diabetic  Discuss these effects in brain, liver, muscle and adipose tissues.  (20 points)

To understand the differences n metabolism between a diabetic and a normal person, we must first understand the physiological role of insulin and how normal metabolism is impaired due to insulin deficiency. To start, immediately after a high-carbohydrate meal, insulin triggers the uptake, storage and utilization of glucose by nearly all tissues of the body (especially muscles, adipose, and liver). In the liver, it activates liver phosphorylase which inhibits glycogen breakdown and glycogen synthase which synthesizes glycogen it also enhances the uptake of glucose by the liver from the plasma by activating glucokinase. When glucose is excess, insulin causes the conversion of glucose into fatty acids (it does this by triggering the generation of pyruvate from glucose via the glycolytic pathway, pyruvate is subsequently converted to acety-CoA, which is a precursor for fatty acid synthesis). The synthesized fatty acids are subsequently packaged as triglycerides in VLDL transported to adipose tissue. Further, it inhibits hormone sensitive lipase in adipose tissue, gluconeogenesis and also decreases the release of amino acids from muscle and other extra-hepatic tissues (pp. 692).

In uncontrolled case of diabetes, the absence of insulin triggers a biochemical starvation mode despite a high concentration of blood glucose. Because of insulin deficiency, the transport of glucose into cells is impaired.


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