Principles of Medical Biochemistry

Southern Blotting determines the size of restriction fragments 167 DNA can be amplified by polymerase chain reaction 168 PCR is used for preimplantation genetic diagnosis 169 Allelic heterogeneity is the biggest challenge for molecular. INTRODUCTION TO METABOLIC PATHWAYS 351 Alternative substrates can be oxidized in the body 351 Metabolic processes are divided 352. Hyperammonemia can be treated with diet and drugs 467 Some amino acids are closely related to common metabolic ones.

Lysine and tryptophan have long catabolic pathways 480 The liver is the most important amino acid organ.

INTRODUCTION TO BIOMOLECULES Chapter 2

INTRODUCTION TO PROTEIN STRUCTURE Chapter 3

OXYGEN BINDING PROTEINS: HEMOGLOBIN AND MYOGLOBIN Chapter 4

ENZYMATIC REACTIONS Chapter 5

COENZYMES

PRINCIPLES OF

MOLECULAR STRUCTURE AND FUNCTION

Part ONE

Biochemistry deals with the molecular function of the body, and the first question we must ask is about the molecular composition of the normal human body. Triglyceride (aka fat) is the main storage form of metabolic energy, found mainly in adipose tissue. Carbohydrates, in the form of glucose and the storage polysaccharide glycogen, are substrates for the generation of metabolic energy.

INTRODUCTION TO BIOMOLECULES

Adenine

• The molecule shown here (2,3-
• Sulfhydryl groups
• Alcoholic hydroxyl groups C. Hydrocarbon groups
• The molecule shown here is acetylsalicylic acid (aspirin). What kind of electrical charge does
• Negatively charged in the stomach; positively charged in the intestine
• Negatively charged both in the stomach and the intestine
• Uncharged in the stomach; negatively charged in the intestine
• Uncharged both in the stomach and the intestine E. Uncharged in the stomach; positively charged in
• Inorganic phosphate, which is a major anion in the intracellular space, has three acidic
• Phosphate molecules absorb protons when the pH decreases during anaerobic exercise
• On average, the phosphate molecules carry more negative charges during anaerobic contraction
• Phosphate molecules release protons when the pH decreases during anaerobic exercise
• The most abundant form of the phosphate molecule in the resting muscle fiber carries one

Ionizable groups also participate in maintaining a constant pH in cells and body fluids through their buffering capacity. What kind of electrical charge does aspirin carry in the stomach at a pH of 2 and in the small intestine at a pH of 7. The most abundant form of the phosphate molecule in the resting muscle fiber carries a molecule in the resting muscle fiber carries a negative charge.

Nucleic acids are the storehouses of genetic information and play several roles in the expression of this information.

INTRODUCTION TO PROTEIN STRUCTURE

• The component of a water-soluble globular protein that is most likely to be present in
• A glutamate side chain B. A histidine side chain
• A phosphate group covalently linked to a serine side chain
• An oligosaccharide covalently linked to an asparagine side chain
• The following structure is an oligopeptide that is acetylated at its amino end and amidated at
• Human blood plasma contains about 7%
• Boiling the serum for 5 minutes
• Adding sodium chloride to a concentration of 35%
• Adjusting the pH to 4.5
• Mixing one volume of plasma with two volumes of pure alcohol
• A genetic engineer wants to produce athletes with increased hemoglobin concentration in
• Your grandmother has become increasingly forgetful during the past 2 years. Last week
• Reduces the synthesis of transthyretin B. Reduces the formation of immunoglobulins
• Serum amyloid A protein B. β 2 -microglobulin
• Chapter 3

It is thought to contribute to β-cell "burnout" in the late stages of the disease. CJD develops when the normal PrPC folds itself into the abnormal PrPSc (Sc stands for "scrapie", the corresponding disease in sheep). The disease was known as kuru after the local word for "trembling". Victims developed tremors, became unable to walk, sometimes laughed compulsively, and died within 1 year of the onset of symptoms.

None of the functional groups in the common amino acids can reversibly bind molecular oxygen.

OXYGEN-BINDING PROTEINS

Without oxygen-binding proteins, the 8,000 L of blood pumped by the heart to the tissues each day could provide only about 30 g of oxygen, or 6% of the total requirement. To remedy this problem, human blood contains 150 g of the oxygen-binding protein hemoglobin per liter, all in erythrocytes. The organic part of the heme group that holds the iron in the center is called protoporphyrin IX (Figure 3.1).

The iron is bound to the free electron pairs of the nitrogen atoms in the four pyrrole rings.

HEMOGLOBIN AND MYOGLOBIN

• A pharmaceutical company is trying to develop a drug that improves tissue oxygenation by
• Binds to the heme iron
• Inhibits the degradation of 2,3-BPG, thereby increasing its concentration in erythrocytes
• Binds to the R conformation of hemoglobin but not the T conformation
• A worker in a chemical factory loses
• Competitive inhibition of oxygen binding B. Oxidation of the heme iron to the ferric state
• Inhibition of hemoglobin synthesis
• The oxygen-binding curve of hemoglobin is sigmoidal because
• The binding of oxygen to a heme group increases the oxygen affinities of the other
• The distal histidine allows the hemoglobin molecule to change its conformation in
• The subunits are held in place by interchain disulfide bonds
• The solubility of the hemoglobin molecule changes with its oxidation state
• Chapter 4

The oxygen binding curve describes the fractional saturation of the heme groups at varying oxygen partial pressures. Oxygen binding to a heme group in hemoglobin increases the oxygen affinities of the remaining heme groups. Oxidation of the heme iron in hemoglobin to the ferric state produces methemoglobin, which is useless for oxygen transport.

Their direction is determined by the concentrations of the substances involved in the extrapulmonary tissues (A) and in the pulmonary capillaries (B).

ENZYMATIC REACTIONS

• During a drug screening program, you find a chemical that decreases the activity of the
• Definitely a competitive inhibitor B. Definitely a noncompetitive inhibitor
• Either a competitive or an irreversible inhibitor E. Either a noncompetitive or an irreversible
• The irreversible enzymatic reaction
• An irreversible inhibitor reacting with the oxaloacetate binding site of the enzyme
• A competitive inhibitor binding to the acetyl-CoA binding site of the enzyme
• A competitive inhibitor binding to the oxaloacetate binding site of the enzyme
• A noncompetitive inhibitor binding to the acetyl- CoA binding site of the enzyme
• A noncompetitive inhibitor binding to the oxaloacetate binding site of the enzyme
• Protonated glutamate and deprotonated aspartate B. Protonated histidine and deprotonated lysine
• Protonated arginine and deprotonated lysine E. Protonated cysteine and deprotonated histidine
• A biotechnology company has cloned four different forms of the enzyme money
• Enzyme 1 B. Enzyme 2
• Which of the four forms of money synthetase is fastest at a saturating ATP
• If the money synthetase reaction is freely reversible, which of the following
• Decreasing the pH value
• Adding another enzyme that destroys ADP C. Using a very low concentration of ATP
• Adding a noncompetitive inhibitorOxaloacetate Acetyl coenzyme A CoA
• Chapter 5

In the enzyme-substrate complex, the substrate binds noncovalently to the active site on the surface of the enzyme protein. The formation of the transition state is the rate-limiting step in the overall reaction. The enzyme facilitates the reaction by lowering the free energy content of the transition state.

Because the reaction rate is proportional to the concentration of the enzyme-substrate complex [equation (18)], Km is also the substrate concentration at which the reaction rate is half-maximal.

G +ATP

Acetyl-CoA

Palmitoyl-CoA

• ADENOSYL METHIONINE DONATES METHYL GROUPS
• Protein kinases are enzymes that phosphorylate amino acid side chains of
• Oxidoreductase B. Hydrolase
• Cyanide is a potent inhibitor of cell respiration that prevents the oxidation of all nutrients
• Heme groups B. FADH 2
• The reaction

This occurs, for example, during acetylation reactions (the “A” in “coenzyme A” stands for “acetylation”) and in the synthesis of triglycerides (see Chapter 25). In most cases, the methyl group donor is S-adenosyl methionine (SAM) (Figure 5.8), which is synthesized in the body from ATP and the amino acid methionine. Some enzymes contain a transition metal such as iron, zinc, copper or manganese in their active site.

This occurs in many oxygenase reactions when ferrous iron (Fe2+) or monovalent copper (Cu+) binds molecular oxygen in the active site of the enzyme. In this case, the electron density on the oxygen of the water molecule increases by This makes water more reactive for nucleophilic attack on carbon by CO2.

Some coenzymes are tightly bound to the enzyme as prosthetic groups, while others are soluble cosubstrates. More important coenzymes include ATP for energy-dependent reactions; NAD, NADP, FAD and FMN for hydrogen transfers; coenzyme A for the activation of organic acids; and SAM for methylation reactions. Some enzymes catalyze their reaction with the help of a metal ion in their active site.

Protein kinases are enzymes that phosphorylate the side chains of amino acids phosphorylate the side chains of amino acids.

DNA, RNA, AND PROTEIN SYNTHESIS Chapter 7

THE HUMAN GENOME Chapter 8

PROTEIN TARGETING AND PROTEOSTASIS Chapter 9

INTRODUCTION TO GENETIC DISEASES Chapter 10

VIRUSES Chapter 11

DNA TECHNOLOGY

GENETIC INFORMATION

DNA, RNA, AND

PROTEIN SYNTHESIS

Part TWO

Transcription is the synthesis of a messenger RNA (mRNA) molecule in the nucleus. The mRNA is the

Translation is the synthesis of a polypeptide by the ribosome, guided by the base sequence of the mRNA. A single gene can be transcribed into thousands of mRNA molecules, and each mRNA can be translated into thousands of polypeptides. For example, red blood cells contain 5 × 108 copies of the β-chain of hemoglobin, but the precursors of red blood cells that make hemoglobin have only two copies of the β-chain gene.

DNA, RNA, AND PROTEIN SYNTHESIS

The high fidelity of DNA replication in E. coli would not be possible without

The high processivity of DNA polymerase III B. The S subunit of DNA polymerase I

The extremely high accuracy of the aminoacyl- tRNA synthetases

• Stop codons are present on

The coding strand of DNA, where they signal the end of transcription

The template strand of DNA, where they signal the end of transcription

The mRNA, where they signal the end of translation

The tRNA, where they signal the end of translation

Termination factors, where they signal the end of translation

A disruption of codon-anticodon pairing during protein synthesis

Premature chain termination during ribosomal protein synthesis

Impaired initiation of ribosomal protein synthesis

Inability of the aminoacyl-tRNA to bind to the ribosome

A change in the genetic code

• Like lactose, the pentose sugar arabinose can be used as a source of metabolic energy

Enzymes of arabinose catabolism are induced when glucose is plentiful

A repressor protein prevents the synthesis of arabinose-catabolizing enzymes in the absence

The cAMP receptor protein prevents the synthesis of arabinose-catabolizing enzymes in

Arabinose acts as a corepressor that is required for the binding of the repressor to the operator

THE HUMAN GENOME

The reason why a fusion of two oocytes in the test tube cannot produce a viable child is

Some imprinted genes would not be expressed C. Transposons would become activated

Female-expressed genes would not be suppressed by male-derived miRNAs

Telomeres would shorten excessively, leading to early senility and death in utero

• Some pharmaceutical companies are trying frantically to find inhibitors of telomerase

Boost the synthesis of muscle proteins B. Prevent viral infections

Cure cancer

Cure acquired immunodeficiency syndrome (AIDS)

Make people immortal

• Alu sequences can cause diseases by jumping into new genomic locations. Their mobility

Transposase and RNA polymerase B. DNA polymerase and RNA replicase

RNA polymerase and reverse transcriptase 4. The report you have just received from the

A sequence in the centromeric DNA

A piece of chromatin attached to the short arm of an acrocentric chromosome

A tandem repeat of variable length

A nonfunctional copy of a gene

A small RNA that regulates gene expression by targeting mRNAs

• Eukaryotic enhancers are

Regulatory DNA sequences within the coding sequences of genes that affect the rate of

Binding sites for general transcription factors in the promoter

Proteins that bind to regulatory base sequences in DNA

DNA sequences outside the promoter region that contain multiple binding sites for regulatory proteins

• In the year 2045, the Surgeon General determines that reverse transcriptase is

DNA transposons and Alu sequences

Acetylation of histones and enhanced transcription

Repression of retroposition by mobile elements

PROTEIN TARGETING AND PROTEOSTASIS

• cell disease is a rare, recessively inherited disease in which one of the enzymes for the attachment of
• A signal sequence has to be expected in the precursors of all the following proteins except
• The sodium-potassium ATPase in the plasma membrane
• Collagen in the extracellular matrix of connective tissues
• Signal peptidase
• Acid maltase, a lysosomal hydrolase
• The deficiency of a ubiquitin ligase can potentially result in
• Abnormal accumulation of ubiquitin in the cell B. Failure to direct lysosomal proteins to the
• Excessive breakdown of some classes of proteins D. Buildup of abnormal proteins in the cells
• Increased mutation rate

Glycosylation in the ER and Golgi apparatus is a complex process requiring at least 400 proteins (2% of the translated genome). In the ER and Golgi apparatus, exoglycosidases remove glucose residues and one or more of the mannose residues from the protein-bound oligosaccharide. This lipid is used as a carrier of the basic oligosaccharide in the endoplasmic reticulum (ER) membrane.

The lysosomal enzymes are synthesized at the rough ER and are glycosylated in the ER and Golgi apparatus. The substrate of the missing enzyme accumulates in the cell to a point where it impairs normal cellular function. Clinical Example 8.3 shows that it is also involved in the elimination of bacteria that have entered the cell.

HSP90 is mainly concerned with the late stages in the folding of newly synthesized proteins. The number in the names of the heat shock proteins indicates their molecular weight. Crohn's disease has long been attributed to an aberrant immune response to components of the normal bacterial flora in the gut.

The formation of these abnormal protein aggregates is favored by age-related decline in the efficiency of the proteostasis system. Misfolded proteins are marked for destruction by the covalent attachment of the small polypeptide ubiquitin, followed by degradation in the proteasome.

INTRODUCTION TO GENETIC DISEASES

• Single-gene disorders, also known as Mendelian disor- ders because of their predictable inheritance patterns,
• You examine a 10-month-old infant who has numerous scaly and ulcerative skin
• Postreplication mismatch repair B. Repair of DNA double-strand breaks
• Nucleotide excision repair
• The most important type of DNA damage in the child described in Question 1 is
• DNA double-strand breaks B. Pyrimidine dimers
• Replication errors D. Insertions and deletions
• An 18-month-old girl of Middle Eastern ancestry, who initially was treated for
• A DNA repair defect C. α -Thalassemia major
• Some patients with sickle cell disease have relatively mild symptoms because they also
• Bone marrow depression B. Elevated β -chain synthesis
• In Europe and North America, the average age at reproduction has increased by several years
• Aneuploidy in both advanced maternal and paternal age
• Maternal age: aneuploidy; paternal age: point mutations
• Maternal age: chromosomal rearrangements;
• Maternal age: copy number changes; paternal age: aneuploidy
• Point mutations in both advanced maternal and paternal age
• A medical student is told by his clinical instructor that genetic diseases are
• Pleiotropy
• Paternal age effect
• Mutation-selection balance D. Heterozygote advantage

DNA variants that are present in all of the child's cells but are not present in either parent are new mutations that arose in the germline of one of the parents. Note that the two thymine residues are in the same strand of the double helix. Others (XPB, XPD) are helicases that separate the strands to the left and right of the lesion.

The "XP" in the names of many repair proteins stands for xeroderma pigmentosum, a disease caused by defects in excision repair proteins. The disease is caused by a deficiency of the ATM (ataxia-telangiectasia mutated) kinase, which is recruited to DNA double-strand breaks where it is activated. Its most important effect is an increase in the synthesis of fetal hemoglobin (HbF), which normally accounts for less than 2% of total hemoglobin in adults.

CO reduces sickling by converting some deoxy-HbS to non-sickled CO-HbS. Most of the unpaired α-chains present in β-thalassemia patients are degraded, but those that survive form abnormal aggregates in cells. In these patients, the severity of the disease is inversely proportional to the HbF level.

In some cases of HPFH without deletion, the condition can be traced to a point mutation in the promoter region of one of the γ-chain genes. These include missense mutations in the genes for β-amyloid precursor protein (APP) and for two subunits (presenilin 1 and 2) of the γ-secretase, which produces the pathogenic β-amyloid by cleaving APP.

VIRUSES

In order to infect a new host cell, a virus has to bind specifically to a “virus receptor” on

In the case of the virus that causes AIDS (retrovirus), this initial interaction involves the virus.

Spike proteins B. Reverse transcriptase

Integrase

• An inhibitor of reverse transcriptase would be useful to

Prevent lytic infection by T4 bacteriophage C. Inhibit homologous recombination between two

Chapter 11

• TALENs (transcription activator like effector nu- cleases) are similar to zinc finger nucleases. They
• To sequence a piece of DNA with the dideoxy method, you will probably want to use
• A pair of primers B. Reverse transcriptase
• You have been instructed by the U.S
• Linkage analysis with closely linked RSPs or SNPs D. cDNA microarrays
• Dot blotting
• Retroviral vectors are more popular for somatic gene therapy than other viral vectors
• They replicate faster than most other viruses B. They contain several copies of their DNA
• They can integrate themselves into the host cell DNA
• Their replication is more accurate than that of most other viruses
• Their DNA has extensive sequence homology with normal cellular DNA
• The steps of classic Southern blotting include (1) denaturation of DNA with alkali,
• A child is born with multiple congenital
• Southern blotting with allele-specific oligonucleotide probes
• Dot blotting
• Cleavage of loxP sites with Cre recombinase D. Array comparative genome hybridization
• PCR-based procedures are often preferred over Southern blotting for the prenatal diagnosis
• PCR requires less DNA; therefore, lengthy cell culturing may not be necessary
• PCR requires less technical skill than Southern blotting and therefore is less costly
• PCR is less sensitive to contamination by extraneous DNA and therefore is less prone to
• Unlike Southern blotting, PCR does not require DNA from many family members
• Unlike Southern blotting, PCR does not require any knowledge of the DNA sequence in and
• The classic PCR procedure (without probes) can be used to
• Amplify the whole dystrophin gene with its 79 exons and 78 introns in one piece
• Diagnose the sickle cell mutation after amniocentesis
• Diagnose HIV infection in people with risky lifestyles
• Detect point mutations in a gene whose sequence is unknown
• Perform all of the above
• In order to genotype a skin color gene in DNA from a 30,000-year-old Neanderthal skeleton,
• Southern blotting with allele-specific probes B. In situ hybridization
• Oligonucleotide microarrays
• Linkage with closely linked microsatellite polymorphisms
• Linkage studies with closely linked microsatellite polymorphisms are the
• There is allelic heterogeneity, and it is not known which mutation in a known disease
• There is locus heterogeneity, and it is not known which gene is mutated in an affected
• No case of the disease has so far occurred in the family
• Population-wide screening is intended
• The exact molecular nature of the mutation is known
• In order to compare the expression of a large number of genes in rhabdomyosarcoma cells
• PCR with nested primers B. Dot blotting
• Cloning in bacteria D. Southern blotting
• Alternatively, the mRNAs from the tumor and normal cells can be compared using

Instead of the mRNA, a complementary DNA (cDNA) probe prepared from the mRNA by reverse transcriptase can be used. Each of the four dideoxynucleotides (ddATP, ddCTP, ddGTP, ddTTP) is labeled with a different fluorescent label. The main problem is the delivery of foreign DNA to the nucleus of the targeted cells.

Viral proteins that disrupt the endosome membrane can also be added to prevent routing of the DNA to the lysosomes. Four of the five patients were cured with chemotherapy, but one succumbed to the leukemia. In these children, the retroviral vector was inserted next to the promoter of the LMO2 gene.

Nucleases and donor DNA must be transferred into the nucleus of target cells. One of the RNA strands binds to the Ago2 protein in the RNA-induced silencing complex (RISC), which then cleaves the target mRNA (see Chapter 7). After fertilization in a test tube, the resulting embryo is tested for the presence of the desired genetic change.

After the gene has been introduced into the germ line, the cellulase will be secreted by the pancreas. A strain of mice is created with loxP sites ( ) flanking one of the exons (exon 4) of the insulin receptor gene.