Biochem Exam 3 Questions with Answers Latest 2024/2025 Verified Updates, Exams of Biochemistry

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1. -isolate protein

-determine partial amino acid sequence of the protein -synthesize oligonucleotides that correspond to portions of the amino acid sequence -use oligonucleotides that correspond to portions of the amino acid sequence -sequence isolated gene: from protein to gene

2. -isolate genomic clone corresponding to an altered trait in mutants

-use genomic DNA to isolate a cDNA for the mRNA encoded by the gene -sequence the cDNA to deduce amino acid sequence of the encoded protein -compare deduced amino acid sequence with that of known proteins to gain insight into function of the protein -use expression vector to produce the encoded protien: from gene to protein

3. -they cleave DNA phosphodiester bonds at specific sequences

-common in bacteria (eliminates infectious viral DNA) -some make staggered cuts (sticky ends) and some make straight cuts (blunt ends): restriction endonucleases

4. -unpaired bases on the ends

-due to endonucleases making staggered cuts -can base pair with each other or complementary sticky ends: sticky ends

5. -no unpaired bases on the ends

-due to endonucleases making straight cuts: blunt ends

6. -restriction enzyme recognition sites are palindromes and exhibit twofold rotational symmetry

-some restriction enzymes can generate cohesive ends that have either 5' (Hind III) or 3' overlaps -other restriction enzymes (Bal I) cut in the center of the symmetrical recog- nition sequence and generate ends with no overlapping cohesive sequences but blunt ends -scissors indicate the cut positions for the enzymes shown: restriction endonu- cleases

7. BamHI, Sau3AI, EcoRI, HindIII, and Notl produce sticky ends

Smal produces blunt ends: examples of restriction endonucleases

8. -cleaves DNA molecules at specific base sequences

-enzyme used in recombinant DNA technology: Type 2 restriction endonucleas- es

9. joints two DNA molecules or fragments

enzyme used in recombinant DNA technology: DNA ligase

10. fills gaps in duplexes by stepwise addition of nucleotides to 3' ends

enzyme used in recombinant DNA technology: DNA polymerase I (E. coli)

11. makes a DNA copy of an RNA molecule

enzyme used in recombinant DNA technology: reverse transcriptase

12. adds a phosphate to the 5'-OH end of a polynucleotide to label it or to permit ligation

enzyme used in recombinant DNA technology: polynucleotide kinase

13. adds homopolymer tails to the 3'-OH ends of a linear duplex

enzyme used in recombinant DNA technology: terminal transferase

14. removes nucleotide residues from the 3' ends of a DNA strand

enzyme used in recombinant DNA technology: exonuclease III

15. removes nucleotides from the 5' ends of a duplex to expose single-strand- ed 3' ends

enzyme used in recombinant DNA technology: bacteriophage (gamma) exonu- clease

16. removes terminal phosphates from the 5' and 3' end (or both)

enzyme used in recombinant DNA technology: alkaline phosphatase

17. -obtain the DNA segment to be cloned

-select a small molecule of DNA capable of autonomous replication -join the two DNA fragments covalently -move the recombinant DNA from the test tube to the host organism -select or identify host cells that contain the recombinant DNA: process of DNA cloning

18. -recombinant DNA is made from vector and target DNAs by annealing and ligation

-the recombinant DNA molecule is introduced into the host cell by transfor- mation or transfection -propagation of the molecule takes place in the host cell -the presence of a vector is typically detected by the antibiotic resistance that is conferred to the host cell -the target DNA is specifically selected by hybridization -cDNA=complementary DNA: basic cloning strategy

19. aka restriction enzymes

recognize and cleave DNA at specific at specific sequences (recognition sequences or restriction sites): restriction endonucleases pt

20.catalyze methylation of host DNA to protect it from digestion by the host cell's restriction

endonucleases: methylases

21.the restriction endonuclease and the corresponding methylase: restric- tion-modification

system

22. enzyme that covalently joins two DNA fragments

-normally functions in DNA repair -human DNA ligase uses ATP -bacterial DNA ligase uses NAD: DNA ligase

23. -cleave with EcoRI restriction enzyme

-anneal DNA fragments and rejoin with DNA ligase: EcoRI recombinant

24. -plasmids

-bacterial artificial chromosomes -yeast artificial chromosomes

cloning vectors allow amplification of inserted DNA segments: 3 popular cloning vectors

25. -a relatively low molecular weight to accommodate larger fragments

-multiple cloning sites useful in cloning a variety of restriction enzyme gener- ated fragments -multiple selectable markers to aid in selecting bacteria with recombinant DNA molecules -an origin of replication -and a high rate of replication: desirable features of a plasmid vector include:

26.bacteriophages, cosmids, yeast artificial chromosomes (YACS): other cloning vectors

27. plasmid: 20

bacteriophage: 25 cosmid: 45 P1 vector: 100 YAC (yeast artificial chromosome): 1000 BAC (bacterial artificial chromosome): 100-300: maximum size of DNA that can be cloned in vectors

28. -derived from a naturally occurring plasmid

-has unique restriction enzyme cleavage sites for insertion of foreign DNA (100bp to 10kb) -has antibiotic resistance genes for selection of transformants containing the plasmid (insertion of DNA at these sites will results in the bacteria becoming sensitive to that antibiotic) -has origin of DNA replication (ori) for propagation in E.coli: structure of pBR322 - a common cloning vector

29. -a commonly used plasmid cloning vector in E.coli

-the molecule is a small double-stranded circle, 2686 base pairs in length, and has a high copy number -pUC19 carries a 54 base-pair multiple cloning site polylinker that contains unique sites for 13 different hexanucleotide-specific restriction endonucleas- es: pUC

30. -composed of the plasmid vector and large segments of cloned DNA

-plasmid vectors developed to allow the cloning of very long segments of DNA (100,00-300,000 bp): bacterial artificial chromosomes (BACs)

31. -composed of the plasmid vector and large segments of cloned DNA

-DNA cloned in a YAC can be altered to study: the function of specialized sequences in chromosome metabolism and mechanisms of gene regulation and expression: yeast artificial chromosomes (YACs)

32. plasmids that can be propagated in cells of 2+ species

incorporate multiple replication origins or other elements: shuttle vectors

33. -bacteria are transformed with the recombinant plasmid

-colonies that grow in tetracycline, but not in ampicillin are isolated -human DNA cut with Pstl -pBR322 DNA cut with Pstl inactivating the ampR gene: cloning a segment of DNA into a plasmid vector

34. -antibiotics, such as penicillin and ampicillin, kill bacteria

-plasmids can carry genes that give a host bacterium a resistance against antibiotics -allows growth (selection) of bacteria that have taken up the plasmid: antibiotic selection

35. -pBR322 is cleaved at the ampR elements by Pstl (Pstl restriction endonu- clease)

-DNA fragments to be cloned are ligated to cleaved pBR322. where ligation is successful, the ampR gene is disrupted. the tetR gene remains intact (DNA ligase) -E.coli cells are transformed, then grown on agar plates containing tetracy- cline to select for those that have taken up plasmid (transformation of E.coli cells) -individual colonies are transferred to matching positions on additional plates. one plate contain t: antibioticR selection

36. -mRNA can be extracted from eukaryotic cells

-all mRNA molecules have a polyA tail: helps in purification of mRNA and serves as a universal template -a DNA strand can be synthesized using mRNA as a template -this is catalyzed by the reverse transcriptase -the end result is a hybrid where the DNA strand is complementary to the mRNA -the hybrid can be converted to duplex DNA, known as cDNA: construction of cDNA

37. mRNA template is annealed to synthetic ologonucleotide (oligo dT) primer

reverse transcriptase and dNTPs yield a complementary DNA strand (mRNA-DNA hybrid) -

reverse transcribes RNA into cDNA mRNA is degraded with alkali - remove RNA with alkali and add poly(dG) tail DNA polymerase I and dNTPs yield double stranded DNA (duplex DNA) from a single stranded cDNA by hybridizing with oligo-dC primer and synthesize complementary strand to give double stranded cDNA -double stranded cDNA gets protec: cDNA formation

38.contains representative copies of cellular mRNA sewuences: cDNA library

39. -can detect a specific DNA fragment in a complex mixture of restriction fragments

-transfer of DNA from gel to a solid matrix -DNA finger printing (RFLP) -genetic counseling (cystic fibrosis) -DNA mapping -sequence homology -gives info about the presence or absence of a particular gene -gives NO information about whether the genes are expressed or not in a given tissue: southern blot technique

40. -unlabeled DNA cut with a restriction nuclease

-labeled DNA of known sizes as size markers -DNA fragments separated by agarose gel electrophoresis -separated DNA fragments blotted onto nitrocellulose paper -remove nitrocellulose paper with tightly bound DNA -labelled DNA probe hybridize to separated DNA -labeled DNA probe hybridized to complementary DNA bands visualized by autoradiography: southern blot: gel transfer

41. -transfer of RNA from a gel to a solid matrix

-gives information about whether RNAs are present in a tissue -often used to determine whether particular genes are expressed -does not give information about whether the gene is present in other tissues-

: northern blotting

42. -transfer of proteins from a gel to a solid matrix

-gives information about the expression of a given protein -labeled antibodies are used as probes: western blotting

43. -we want to study the protein product of the gene

-special plasmids called expression vectors contain sequences that allow transcription of the inserted gene: expression of cloned genes

44. they have:

-promoter sequences -operator sequences -code for ribosome binding site -transcription termination sequences: how do expression vectors differ from cloning vectors

45.some eukaryotic proteins can be produced in E.coli cells from plasmid vectors containing the lac

promoter: eukaryotic protein production & lac promoter

46. used to amplify DNA in the test tube:

-can amplify regions of interest (genes) within linear DNA -can amplify complete circular plasmids: use of polymerase chain reaction PCR

47. mix together:

-target DNA -primers (oligonucelotides complementary to target) -nucleotides: dATP, dCTP, dGTP, dTTP -thermostable DNA polymerase place the mixture into a thermocycler: -melt DNA at about 95 C -cool separated strands to about 50-60 C -primers anneal to the target -polymerase extends primers in the 5'-3' direction -after a round of elongation is done, repeat the steps: PCR process

48. -add excess primers 1 and 2 , dNTPS, and Taq polymerase (extend primers with Taq polymerase)

-heat to 95 C to melt strands

-cool to 60 to anneal primers -add thermostable DNA polymerase to catalyze 5'-3' DNA synthesis -after 25 cycles, the target sequence has been amplified about 10^6 fold -after cooling to anneal, repeat this process: PCR process pt

49. -find cleavage sites for Mst II (cleavage by Mst II)

-denaturation to single strands -hybridization with specific probe -produces 1.1-Kb fragment: Normal beta gene

50. missing cleavage site

1.3-Kb fragment (extra 0.2Kb): sickle cell gene

51. -denature DNA, and transfer to nylon membrane

-incubate with probe, then wash (radiolabeled DNA probe) -expose xray film to membrane: the CSI effect

52. -Molecule Subjected to Electrophoresis: DNA

-Probe Molecule: DNA: molecule subjected to electrophoresis and probe molecule for southern blotting

53. Process:

-DNA from a sample is first digested with restriction enzymes, which cut the unlabeled DNA at specific recognition sequences. -The resulting DNA fragments are separated by size using agarose gel elec- trophoresis. The smaller fragments migrate faster and end up nearer to the positive electrode. -After electrophoresis, the gel is soaked in a denaturing solution (usually an alkaline solution) to depurinate the DNA, making it single-stranded. -The DNA in the gel is then transferred onto a ni: process of sourthern blotting (better explained version)

54. -Molecule Subjected to Electrophoresis: RNA

-Probe Molecule: DNA or RNA: molecule subjected to electrophoresis and probe molecule for northern blotting

55. Process:

-RNA from a sample is separated by size using gel electrophoresis. It is usually denatured before loading onto the gel. -The separated RNA is then transferred onto a membrane (usually nylon or nitrocellulose) by capillary or electroblotting. -The membrane is hybridized with a labeled DNA or RNA probe that is com- plementary to the target RNA sequence. -Unbound probe is washed away, and the membrane is exposed to X-ray film or a phosphorimager to visualize the labeled RNA bands.: process of northern blotting (better explained)

56. -Molecule Subjected to Electrophoresis: Protein

-Probe Molecule: Antibodies (or sometimes ligands for specific proteins): mol- ecule subjected to electrophoresis and probe molecule for western blotting

57. Process:

-Proteins from a sample are separated by size using gel electrophoresis (usually SDS-PAGE, which denatures and uniformly charges the proteins). -After electrophoresis, the separated proteins are transferred from the gel onto a membrane (usually nitrocellulose or PVDF) by capillary or electroblotting. -The membrane is then blocked with a protein (like bovine serum albumin or non-fat milk) to prevent non-specific binding of antibodies. -The membrane is probed with specific antibodies that: process of western blotting (better explained)

58.Sanger dideoxy sequencing, often referred to simply as Sanger sequenc- ing, is a widely used

method for determining the sequence of nucleotides (A, T, C, and G) in a DNA molecule.: use for sanger dideoxy sequencing

59. DNA Template Preparation:

-A segment of DNA that you want to sequence is selected. This DNA segment serves as the template for the sequencing reaction. Primer Annealing: -A short piece of single-stranded DNA, known as a primer, is designed to be complementary to a specific region of the template DNA. The primer is typically labeled with a fluorescent or radioactive marker for detection.: first two steps of sanger dideoxy sequencing (preparation and primer annealing)

60. DNA Synthesis Reaction:

-A DNA synthesis reaction mixture is prepared, containing:The template DNA.A mixture of regular

deoxynucleotides (dATP, dTTP, dCTP, and dGTP), which are the building blocks of DNA.A small amount of a modified deoxynu- cleotide called a dideoxynucleotide (ddNTP), which lacks a 3' hydroxyl group, preventing further extension of the DNA chain.DNA polymerase enzyme, which catalyzes the synthesis of a new DNA strand. Polymerization: -The DNA polymerase enzyme extends the primer: dna synthesis reaction and polymerization of sanger dideoxy sequencing

61. Termination and Fragmentation:

-When a ddNTP is added to the growing DNA strand, the absence of a 3' hydroxyl group prevents further extension. This terminates the chain at that specific position. -The result is a mixture of DNA fragments, each terminating at a different position in the sequence. Separation by Gel Electrophoresis: -The mixture of DNA fragments is separated by size using gel electrophoresis. A gel is prepared with a porous matrix, and an electric field is applied. Smaller DNA fr: termination/fragmentation, separation, visualization and analysis steps of sanger dideoxy sequencing

62.the gene of interest together with appropriate regulatory elements is mi- croinjected into a

fertilized mouse egg which is then inserted into the uterus of a foster mother mouse: transgenic animals

63. -microarray chips contain fragments from genes in the group to be ana- lyzed

-full genome of bacteria or yeast, or protein-encoding families from larger genomes -mRNA or cDNA from different samples are differentially tagged -analysis on the same chip shows differences: DNA microarrays show differ- ences in gene expression

64. -isolate mRNAs from cells at two stages of development; each mRNA sample represents

all the genes expressed in the cells at that stage -convert mRNAs to cDNAs with reverse transcriptase, using fluorescently labeled deoxyribonucleoside triphosphate -add the cDNAs to a microarray; fluorescent cDNAs anneal to complementary sequences on the microarray -wash and measure green and red fluorescence over each spot -removal of unhybridized probe

-each fluorescent spot represents a gene expressed in the: DNA microarray process

65. -start with double stranded RNA and it interacts with a dicer enzyme to result in an siRNA

(small-interfering RNA) which can silence genes -vector-expressed siRNA is incorporated into a multi-protein nuclease com- plex known as the RNA- induced silencing complex(RISC) -homology between the sense portion of the siRNA sequence and the mRNA enables the nuclease enzyme to bind and cleave the TF-encoding transcript into small pieces -these are then degraded by the cell -once cleavage has occurred, the R: siRNA gene silencing

66. -zinc finger nucleases (ZFNs)

-TALENs (transcription activator-like effector nucleases) -CRISPR/Cas9 (clustered, regularly interspersed, short palindromic: genome editing "tools"

67. -CRISPR stands for clustered, regularly interspaced short palindromic repeats

-CRISPR sequences=regularly spaced short repeats in the bacterial genome, surrounding sequences derived from phage pathogens that previously infect- ed the bacterium -Cas protein=nuclease: CRISPR/Cas systems

68. -guide RNAs: transcribed viral spaced sequences that are cleaved

-trans activating CRISPR RNA (tracrRNA) -1+ Cas proteins -the complex binds and destroys invading bacteriophage DNA by the Cas protein nuclease activities: components of the CRISPR/Cas complex

69.The CRISPR system was initially discovered as a part of the bacterial im- mune system. It

provides bacteria with a defense mechanism against invading viruses by storing a "memory" of viral DNA sequences. When the bacteria encounter the same virus in the future, they can use this memory to recognize and defend against it.: CRISPR discovery

70. requires only two components:

-single Cas protein (Cas9) -single guide RNA (sgRNA): consists of gRNA and tracrRNA fused into a single RNA: current CRISPR technology

71. has two separate nuclease domains:

-each domain cleaves one strand of DNA: Cas9 protein

72. -the sgRNA (single guide RNA) sequence can be altered to target any genomic sequence

-required to pair with target DNA sequences and to activate the nuclease domains: single guide RNA (sgRNA)

73.END OF RECOMBINANT POWERPOINT: END OF RECOMBINANT POWER- POINT

74. -have the formula (CxH2O)n, where n=# of carbon

-hydrates of carbon -ex: glucose c6h12o6, or (cXh20)6 where= -aldehyde or ketone compounds with multiple -OH groups: carbohydrates

75. -Monosaccharides: simplest sugars—glucose, fructose

-Disaccharides: 2 monosaccharide units linked together—maltose, sucrose, lactose -Oligosaccharides: H 3-20 units linked in linear or branched chains -Polysaccharides: 100's of units linked in repeating fashion—glycogen, cellu- lose, chitin (found in arthropod exoskeletons): types of carbohydrates

76. -store energy: glycogen (animals) and starch (plants)

-make up part of structure of DNA and RNA -components of bacterial cell walls and extracellular matrix -sometimes linked to proteins or lipids (glycoconjugates): biological roles of carbohydrates

77. The Trioses:

glyceraldehyde: an aldotriose hidroxyacetone: a ketotriose: the simplest monosaccharides

78.what monosaccharide is this: glyceraldehydde

79.what monosaccharide is this: dihydroxyacetone

80. glyceraldehyde has a chiral carbon:

-therefore, it exists in two enantiomeric forms: D (dextrorotary) and L (levoro- tary) -non superimposable mirror images -rotate plane polarized light in opposite directions -what about a racemic mixture? -D found in sugars fischer projection: -2D representation of a 3D structure: steriochemistry of sugars

81. -molecules whose atoms are connected in the same order, but differ in their spatial arrangement

-number of sterioisomers possible= 2^n, where n= # of chiral carbons -aldotrioses (3): 2^1= -aldotetroses (4): 2^2= -aldopentoses (5): 2^3= -aldohexoses (6): 2^4= -aldoheptoses (7): 2^5= -this # includes enantiomers (both D and L) and diasteromers (sterioisomers that are not mirror images): how many sterioisomers are possible for a sugar containing "n" carbons?

82. -aldoses have an aldehyde group

-ketoses have a ketone group -ketone has one fewer chiral carbons than corresponding aldose: aldoses versus ketoses

83.Hemiacetals, hemiketals, acetals, and ketals are all types of chemical compounds that are

formed through reactions involving alcohols and car- bonyl compounds (aldehydes or ketones).:

Hemiacetals, Hemiketals, Acetals and Ketals

84. -A hemiacetal is formed when an alcohol (R-OH) reacts with an aldehyde (R'-CHO) or a ketone

(R'-C(=O)-R'') in the presence of an acid catalyst. -The result is a molecule with a hydroxyl (-OH) group and an alkoxy group (-OR') attached to the same carbon atom. -Hemiacetals are relatively unstable and can readily convert to their respective carbonyl compounds under mild conditions.: hemiacetal

85. -A hemiketal is formed when an alcohol (R-OH) reacts with a ketone (R'-C(=O)-R'') in the

presence of an acid catalyst. -Similar to hemiacetals, a hemiketal contains a hydroxyl (-OH) group and an alkoxy group (-OR') attached to the same carbon atom. -Like hemiacetals, hemiketals are also relatively unstable and can convert back to their respective carbonyl compounds under mild conditions.: hemike- tals

86. -An acetal is formed when a hemiacetal reacts with another alcohol mole- cule in the presence

of an acid catalyst. -This reaction leads to the formation of a molecule with two alkoxy (-OR') groups attached to the same carbon atom. -Acetals are more stable than hemiacetals and are less likely to revert back to their carbonyl forms.: acetal

87. -A ketal is formed when a hemiketal reacts with another alcohol molecule in the presence of an

acid catalyst. -This leads to the formation of a molecule with two alkoxy (-OR') groups attached to the same carbon atom. -Similar to acetals, ketals are more stable than hemiketals and are less likely to revert back to their carbonyl forms.: ketal

88. -cyclization involved intramolecular nucleophilic attack of penultimate OH on carbonyl (C1)

carbon -hemiacetal is produced: glucose exists in cyclic forms

89. two different anomers are possible, alpha and beta:

-C1 is anomeric carbon -the two forms alpha and beta are in equilibrium via open-chain form -process of interconversion is called mutorotation -for glucose, equilibrium distribution of forms is: 33% alpha 66% beta 1% open-chain: anomers of glucose/hemiacetal: -a-D-glucopyranose -b-D-glucopyranose

90. a useful method for describing the 3D conformations of cyclic structures-

: hawthorn projection

91.cyclic form is a hemiketal: what does the cyclic form of fructose produce

92. -chair conformation is most stable

-all OH and CH2OH groups in equatorial (not axial) positions -avoids steric hinderance -similarly, furanose ring of fructose exists in different conformations -D-ribose (in DNA and RNA) exists in C-3-endo or C-2-endo envelope confor- mations: different conformations possible for cyclic forms of sugars: glucose

93. -all have, or can form a free aldehyde group

-examples: glucose, galactose, mannose, fructose, maltose, and lactose: re- ducing sugars

94.-because a keto-enol tautomerization allows formation of aldehyde from ketone

(ketone=enediol=aldehyde): why is fructose a reducing sugar if it has a ketone?

95. -because its C2 carbon is tied up in forming an acetal linkage

-C5 carbon is not an anomeric carbon, so it isn't a reducing sugar: why is sucrose not a reducing sugar?

96. -contain two monosacch. units connected by an o-glycosidic (acetal) link- age

-they are acetals or ketals examples: -maltose (Glc-a(1,4)-Glc) -lactose (Gal-b(1,4)-Glc)

-sucrose (Glc-a(1,2)-Fru) the o-glycosidic acetal bond can be enzymatically hydrolyzed to release monosacch. sugars -maltose: maltase; sucrose: sucrase; lactose: lactase: disaccharides

97. -although infants are lactose tolerant, many adults are lactose intolerant

-they have deficiency in expression of gene for the enzyme lactase, which is needed to break down lactose -at least 2 separate evolutionary origins of lactase persistence ---> one west- ern european and the other african (both related to dairy industry): lactose (in)tolerance in infants and adults

98. -disacch. performing hydrolysis break down to form monosacch.

-2 monosacch. performing condensation rxn can result in a(1,4)o-glycosidic linkage in maltose -both alpha and beta anomers exist; reducing sugar: maltose: a(1,4)o-glycosidic linkage

99. starch (storage form of glucose in plants) has two components:

-amylose: linear polymer of glucose essentially polymaltose only a(1,4) linkages -amylopectin: branched contains a(1,6) branch points as well as a(1,4)links branches every 30 residues: starch polysaccharide

100. glycogen (storage form of glucose in animals):

-similar to amylopectin, but branch points occur more frequently (every 10 glucose residues) -way of storing energy for quick release, or to maintain blood sugar levels -two forms: muscle and liver: glycogen polysaccharide

101. cellulose

-linear polymer of glucose units connected by b(1,4) linkages -alternating residues are "flipped"; strands linked by hydrogen bonds -high tensile strength: found in woody stems of plants, blades of grass

-hydrolyzed by the enzyme cellulase that humans lack (termites, ruminants have enzyme by virtue of symbiotic bacteria in their guts): cellulose polysac- charide

102. -examples of heteropolysaccharides

-polymer of disaccharide repeating units, either glucosamine or galac- tosamine, having acidic sulfate or carboxylate groups found in the ECM in proteoglycans -complex of carbohydrate and protein -very large -roles: cushioning, lubrication, adhesion: glycosaminoglycans

103. glycoproteins:

-O linked glycosylation of either serine or threonine residues -N linked glycosylation of asparagine residues -residue must exist within a certain -glycosylation on (i) secreted proteins (EPO, LSH, TSH, etc.), and (ii) extracel- lular face of cell surface proteins, and (iii) lysosomal proteins -important for protein stability and acitivity (EPO) proteoglycans: -complexes of proteins and glycosaminoglycans glycolipids: glycoconjugates

104. aggrecan

-has 3 globular domains and an extended domain modified with GAGs CS and KS -MW=2500 kDa -component of cartilage -plays role as shock absorber -globular domains involved in hyaluronan binding, cell adhesion, and chon- drocyte apoptosis: proteoglycans

105. -hyaluronan (up to 50k repeating disacch.)

-aggrecan core protein -chondroitin sulfate -link proteins -keratan sulfate: proteoglycan aggregates

106. -example of staphylococcus aureus, a gram positive bacterium

-contains heteropolymers of alternating b(1,4)-linked MurNAc (N-acetylmu- ramic acid) and GIcNAc (N-acetylglucosamine )units that are cross linked by short peptides -lysozyme hydrolyzes the glycosidic bond between GIcNAs and MurNAc residues: the peptidoglycan layer of the bacterial cell wall

107. -penicillin inhibits the formation of peptide cross links: Which bond does penicillin

prevent formation of in the peptidoglycan layer of the bacterial cell wall?

108. -proteins destined for secretion, the cell surface, or the lysosomes are synthesized by

ribosomes on the rough ER -N linked glycosylation of proteins begins in the ER lumen and continues in the golgi complex -this involved modified polyprenol known as dolichol phosphate -O linked glycosylation occurs only in the golgy -the golgi complex is the majoe sorting center of the cell and signals encoded in the amino acid sequence and #D structures of proteins direct destination- : cellular synthesis and sorting of glycoproteins

109. whole series of enzymes delivered to the wrong destination in the cell, which results in

high levels in blood and urine -normally, the enzymes contain mannose-6-phosphate residue, but in I-Cell disease, mannose remains unphosphorylated -mannose6phosphate is a marker that directs many hydrolytic enzymes from golgi complex to lysosome -consequence is mistargeting of 6 essential enzymes -as a result, lysosomes contain large inclusions of undigested glycosamino- glycans and glycolipids -I cell disease: I-Cell Disease

110. -antigens attached to proteins and lipids on RBC surface

-A, B differ by group attached to O-antigen at 3rd sugar residue (Gal) -specific glycosyltransferases catalyze attachment of the different groups: people with O-type blood lack functional transferases

blood typing important for transfusions: -if wrong type given, body's immune system will recognize as foreign -blood cell lysis results in: hypotension, kideny failure, shock, and even death -Type AB=universal acceptor; Type O=universal dono: blood antigen contain oligosaccharides

111. -In type O blood, there are no A or B antigens present on the surface of red blood cells

-Type O individuals have naturally occurring antibodies in their plasma against both A and B antigens. This means that if type O blood is transfused into a person with type A, B, or AB blood, the antibodies in the recipient's plasma will not react with the donor's red blood cells. -Transfusion reactions occur when there is a mismatch between the antigens on the donor's red blood cells and the antibodies in t: why is type O the universal donor?

112. -In type AB blood, both A and B antigens are present on the surface of red blood cells.

This means that individuals with type AB blood do not have antibodies against A or B antigens in their plasma -Because individuals with type AB blood do not have antibodies against A or B antigens, they do not react against A or B antigens if they are present on donor red blood cells: why is type AB blood universal acceptor?

113. -cell surface proteins that recognize and bind specific carbohydrate structures on

neighboring cells' surface one class of glycan-binding proteins called Lectins: -ex: concanavalin A and agglutinin -mediate cell-cell recognition, cell adhesion, cell signaling and protein target- ing within the cell -each lectin protein contains two or more sites for binding carbohydrate units -interactions act as molecular "velcro" to allow cell-cell adhesion -the mannose-6-phosphate receptor is an intracellular: glycan-binding proteins

114. END OF CARBOHYDRATES LECTURE: END OF CARBOHYDRATES LEC- TURE

115. -He might have galactosemia, possibly Type I (infantile), the most severe form

-There are 3 types of galactosemia (I, II and III), due to deficiency in one of three enzymes involved in galactose metabolism, GALT, GALK, or GALE: Rosa Gonzales brought her 3-day-old baby Juan to the emergency room. Juan looked ill; he was jaundiced and lethargic. Rosa said he had vomited after being breast-fed.

Lab tests showed high levels of galactose in his blood and urine. What might be wrong with Juan?

116. Type I due to Galactose-1-phosphate uridylyl transferase (GALT) deficien- cy

• Autosomal recessive; occurs in 1 in 50,000 newborns
• Other, later, symptoms include an enlarged liver, kidney failure, mental de- terioration and cataracts (due to conversion of galactose to galactitol, an osmotically active compound)
• Galactose-1-phosphate builds up in the liver, causing "phosphate trapping" and liver enlargement. Why?: What causes Type 1 (infantile) galactosemia

117. -This enzyme (GALT-Galactose-1-phosphate) is responsible for convert- ing galactose-1-

phosphate into glucose-1-phosphate, a critical step in the metabolism of galactose -When GALT is deficient or absent, galactose-1-phosphate cannot be effec- tively converted to glucose-1-phosphate. This leads to the accumulation of galactose-1-phosphate in cells, particularly in the liver -Galactose-1-phosphate is a sugar phosphate molecule that contains a high-energy phosphate bond. In normal metabolism, this phos: how does a deficiency in GALT (type 1 galactosemia) cause phosphate trapping?

118. The increased levels of galactose-1-phosphate and the associated phos- phate trapping

can cause cellular stress and damage in the liver. This can lead to hepatomegaly, or liver enlargement, as the liver attempts to cope with the metabolic imbalance and increased metabolic demand: how does type 1 galactosemia cause liver enlargement?

119. types 2 and 3 galactosemia are more rare and generally less severe: type 2 and 3

galactosemia

120. -treatment would involve feeding the baby a milk-product-free diet

-By eliminating milk and dairy products from the diet, individuals with Galac- tosemia avoid the direct ingestion of galactose. This is crucial because galac- tose cannot be properly metabolized in their bodies: treatment of galactosemia

121. -Aerobic energy production refers to the process by which cells generate energy in the

presence of oxygen. -It involves the breakdown of molecules like glucose and other fuels to produce ATP (adenosine triphosphate), which serves as the primary energy currency of cells

phase 1: the oxidation of fuels phase 2: ATP generation from oxidative phosphorylation: Cellular Respiration: Aerobic Energy Production from Glucose and other Fuels

122. -oxidation of glucose to pyruvate (or lactate)

-produce energy for biosynthesis -can occur anaerobically or aerobically -occurs in cytosol -a central and highly regulated pathway: glycolysis

123. pyruvate has at least 2 fates:

-complete oxidation to CO2 -fermentation to lactate: fates of pyruvate in glycolysis

124. 1: conversion of glucose to fructose-1,6-bisphosphate (uses 2 ATPs) 2: cleavage of

fructose-1,6-bisphosphate to DHAP and glyceralde- hyde-3-phosphate (G3P) 3: conversion of G3P to pyruvate (produces 4 ATPs and 2 NADHs): 3 stages of glycolysis

125. sprint:

-low O2 in final seconds of a sprint -Under high-intensity conditions like a sprint, glycolysis operates in an anaer- obic fashion, meaning without the presence of oxygen. As a result, pyruvate is converted into lactate to regenerate NAD+ (nicotinamide adenine dinu- cleotide), allowing glycolysis to continue at a high rate. slow exercise: -normal O2 -With sufficient oxygen available, pyruvate from glycolysis is transported into the mitochondria and enters the citric acid cycle (Krebs cyc: Utilization of Glycolysis in Muscle during a Sprint, and Slower Exercise

126. the only energy source in:

-RBCs -cornea and lens of eye, and certain regions of the retina -renal medulla -testis

-leukocytes -white (fast twitch) muscle fibers glucose is the main fuel for brain aerobic glycolysis is important in tumor cells (warburg effect) and embryonic stem cells in which metabolic shift occurs during implantation: anaerobic glycolysis

127. -overall delta G=23 kcal/mol of glucose under anaerobic conditions

-much less than for aerobic process -lactate is the end product in this case equation: Glucose + 2 NAD+ + 2 ADP + 2 Pi -> 2 Lactate + 2 NADH + 2 ATP + 2 H2O: anaerobic glycolysis

128. Know:

-all steps of pathway (including cofactors) -whether ATP or NADH is consumed or produced at a given step -which steps are irreversible -structures of all intermediates in pathway -substrate level phosphorylation steps: What to know for glycolysis

129. conversion of glucose to glucose-6-phosphate:

-IRREVERSIBLE (equilibrium lies far to right, delta G cellular is -8.0kcal/mol) -hexokinase dramatically illustrates "induced fit" phenomenon: conformation change occurs upon glucose binding (open-closed) -in muscles, allosterically inhibited by Glu-6-P -in liver and pancreatic beta cells, known as glucokinase Glu-6-P has several possible fates: -glycolysis; glycogenesis; ribose-5-phosphate and NADPH production via the pentose phosphate pathway equ: step 1 of glycolysis

130. isomerization of glu-6-P to fructose-6-phosphate

-phosphoglucose isomerase permits this -REVERSIBLE -delta G cellular is -0.6kcal/mol: step 2 of glycolysis

131. -phosphofructokinase (PFK-1) catalyzes the first committed step of gly- colytic pathway

-IRREVERSIBLE STEP

-delta G cellular is -5.3kcal/mol PFK-1 is allosterically regulated -inhibitors: ATP (liver and muscle), H+ (muscle), citrate (liver) -activators: AMP (muscle), Fru-2,6-bisP (liver( -mammalian enzyme arose via gene duplication and divergence from bacter- ial-like ancestral gene equation: Fru-6-P + ATP (with PFK-1) --> Fru-1,6-bisP + ADP + H+: step 3 of glycolysis

132. -aldolase enzyme cleaves fru-1,6-bisP into dihydroxyacetone phosphate (DHAP) and

glyceraldehyde-3-phosphate (GAP) -GAP continues down pathway: step 4 of glycolysis

133. -isomerization of ketose into aldose; intramolecular redox reaction

-converts DHAP into glyceraldehyde-3-phosphate -enzyme is triose phosphate isomerase (TIM) -involved formation of enediol intermediate -REVERSIBLE -delta G cellular = +0.6 kcal/mol -TIM has alpha/beta barrel structure catalytically nearly "perfect" enzyme: -Kcat/Km=2*10^8 m-1s-1 -active site structural triangles suppress side reaction (ex: formation of methyl glyoxal from enediol intermediate) -essential glutamate and histidin: step 5 of glycolysis

134. conversion of GAP into 1,3-bisphosphoglycerate

reaction occurs in 2 stages: -oxidation of -CHO to -CO2 using NAD+ (delta G is -12kcal/mol) and h2O -joining of -CO2 and Pi to form acyl-P product, 1,3-BPG (delta G cellular is +12 kcal/mol) -the two stages must be coupled: favorable oxidation drives unfavorable formation of acyl-P compound -coupling occurs via formation of high energy thioester intermediate involving active site cysteine (Cys-

-overall, 2 NADHs are produced from 2 NAD+s equa: step 6 of glycolysis

135. -phosphoglycerate kinase catalyzes the reaction of 1,3-BPG into 3-phos- phoglycerate

-REVERSIBLE

-delta G is +0.3kcal/mol -SUBSTRATE LEVEL PHOSPHORYLATION OCCURS -generates 2 ATPs equation: 1,3-BPG + ADP + H+ --->3PG + ATP: step 7 of glycolysis

136. phosphoglycerate mutase converts 3PG into 2PG:

-catalyzes intramolecular group transfer -employs active site phospho-histidine residue -catalytic amounts of 2,3-BPG needed to maintain histidine in phosphorylated state -2,3-BPG is synthesized from 1,3-BPG by special mutase -levels of 2,3-BPG low in most cells (but high in RBCs) -REVERSIBLE -delta G cellular = +0.2kcal/mol: step 8 of glycolysis

137. enolase converts 2PG into phosphoenolpyruvate:

-REVERSIBLE

-delta G cellular is -0.8kcal/mol -dehydration of substrate markedly elevates P-group transfer potential -delta G of 2PG= -3kcal/mol -delta G of PEP= -14 kcal/mol: step 9 of glycolysis

138. pyruvate kinase converts phosphoenolpyruvate into pyruvate:

-energy that is trapped by P within unstable enol form is released when enol changes to more stable ketone form upon loss of P: enol-ketone conversion drives reaction -SUBSTRATE LEVEL PHOSPHORYLATION OCCURS -2 more ATPs are produced equation: phosphoenolpyruvate (PEP) + ADP+ H+ ---> pyruvate + ATP:

139. regeneration of NAD+ under anaerobic conditions:

-lactic acid fermentation -occurs under O2 limiting conditions -is necessary to maintain REDOX balance -NADH and NAD+ in limited supply, so regeneration is critical -NAD+ is necessary for G3P DHase step; without NAD+, glycolysis would come to a standstill: step 11 of glycolysis

140. -added to public drinking water because it is a good anticaries agent

-inhibits glycolytic enzyme enolase: inhibition is "quasi-irreversible" -can bind to other enzymes as F-/HF or as AlF4 complex to modulate bacterial metabolism -fluoroapatite (FAP) containing enamel exhibits higher resistance to acid, leading to lower demineralization rate than hydroxyapatite, HAP -decreased adhesion of bacteria such as S.mutans to FAP vs. to HAP: fluoride

141. -allosteric control

-covalent modification via protein phosphorylation and dephosphorylation -different isozymic forms -transcriptional control (enzyme induction) -sequestration by binding another protien -subcellular translocation: modes of regulation of glycolytic enzymes

142. at the irreversible steps catalyzed by the enzymes:

-hexokinase -phosphofructokinase -pyruvate kinase