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H I G H - Y I E L D P R I N C I P L E S I N
Biochemistry
75
Biochemistry is the study of carbon compounds that crawl.
Mike Adams
This high-yield material includes molecular biology, genetics, cell bi-
ology, and principles of metabolism (especially vitamins, cofactors,
minerals, and single-enzyme-deciency diseases). When studying
metabolic pathways, emphasize important regulatory steps and en-
zyme deciencies that result in disease. For example, understanding
the defect in Lesch-Nyhan syndrome and its clinical consequences is
higher yield than memorizing every intermediate in the purine sal-
vage pathway. Do not spend time on hard-core organic chemistry,
mechanisms, and physical chemistry. Detailed chemical structures
are infrequently tested. Familiarity with the latest biochemical tech-
niques that have medical relevancesuch as enzyme-linked im-
munosorbent assay (ELISA), immunoelectrophoresis, Southern blot-
ting, and PCRis useful. Beware if you placed out of your medical
schools biochemistry class, for the emphasis of the test differs from
that of many undergraduate courses. Review the related biochemistry
when studying pharmacology or genetic diseases as a way to reinforce
and integrate the material.
9610_02.2-Biochem 11/9/05 7:23 PM Page 75
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BI OCHEMI STRYHI GH-YI ELD CLI NI CAL VI GNETTES
Full-term neonate of uneventful What is the diagnosis? PKU.
delivery becomes mentally
retarded and hyperactive and
has a musty odor.
Stressed executive comes What is the mechanism? NADH increase prevents
home from work, consumes 7 gluconeogenesis by shunting
or 8 martinis in rapid succession pyruvate and oxaloacetate to
before dinner, and becomes lactate and malate.
hypoglycemic.
2-year-old girl has an in What is the diagnosis? Kwashiorkor.
abdominal girth, failure to thrive,
and skin and hair depigmentation.
Alcoholic develops a rash, What is the vitamin Vitamin B
3
(pellagra).
diarrhea, and altered mental deciency?
status.
51-year-old man has black spots What is the diagnosis? Alkaptonuria.
in his sclera and has noted that
his urine turns black upon
standing.
25-year-old male complains What is the disease, Familial hypercholesterolemia;
of severe chest pain and has and where is the defect? LDL receptor.
xanthomas of his Achilles
tendons.
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BI OCHEMI STRYMOLECULAR
Chromatin Condensed by () charged DNA looped twice Think of beads on a string.
structure around (+) charged H2A, H2B, H3, and H4
histone octamers (nucleosome bead). H1 ties
nucleosomes together in a string (30-nm ber).
In mitosis, DNA condenses to form mitotic
chromosomes.
Heterochromatin Condensed, transcriptionally inactive.
Euchromatin Less condensed, transcriptionally active. Eu = true, truly transcribed.
Nucleotides Purines (A, G) have 2 rings. Pyrimidines (C, T, U) PURe As Gold: PURines.
have 1 ring. Guanine has a ketone. Thymine has CUT the PY (pie):
a methyl. Deamination of cytosine makes uracil. PYrimidines.
Uracil found in RNA; thymine in DNA. THYmine has a meTHYl.
G-C bond (3 H-bonds) stronger than A-T bond
(2 H-bonds). G-C content melting
temperature.
Nucleotides are linked by 3-5 phosphodiesterase bond.
Transition vs. Transitionsubstituting purine for purine or TransItion = Identical type.
transversion pyrimidine for pyrimidine.
Transversionsubstituting purine for pyrimidine TransVersion = conVersion
or vice versa. between types.
Genetic code Unambiguouseach codon species only 1 amino acid.
features Degeneratemore than 1 codon may code for same amino acid.
Commaless, nonoverlapping (except some viruses).
Universal (exceptions include mitochondria, archaeobacteria, Mycoplasma, and some
yeasts).
77
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Aspartate
Purine (A, G) Pyrimidine (C, T, U)
CO
2
Glycine
N
C
N
C
C
C
N
N
C
Glutamine
N
10
Formyl-
tetrahydrofolate
N
10
Formyl-
tetrahydrofolate
N
C
N
C
C
C
Carbamoyl
phosphate
Aspartate
Nucleosome core
histones H2A, H2B, H3, H4
DNA
Histone H1
9610_02.2-Biochem 11/9/05 7:23 PM Page 77
BI OCHEMI STRYMOLECULAR ( cont i nued)
Mutations in DNA Silentsame aa, often base change in 3rd position Severity of damage: nonsense
of codon (tRNA wobble). > missense > silent.
Missensechanged aa (conservativenew aa is
similar in chemical structure).
Nonsensechange resulting in early stop codon. Stop the nonsense!
Frame shiftchange resulting in misreading of all
nucleotides downstream, usually resulting in a
truncated protein.
Prokaryotic DNA Single origin of replicationcontinuous DNA DNA polymerase III has
replication and synthesis on leading strand and discontinuous 5 3 synthesis and
DNA polymerases (Okazaki fragments) on lagging strand. proofreads with 3 5
DNA topoisomerases create a nick in the helix to exonuclease.
relieve supercoils. DNA polymerase I excises
Primase makes an RNA primer on which DNA RNA primer with 5 3
polymerase III can initiate replication. exonuclease.
DNA polymerase III elongates the chain by adding
deoxynucleotides to the 3 end until it reaches
primer of preceding fragment. 3 5
exonuclease activity proofreads each added
nucleotide.
DNA polymerase I degrades RNA primer.
DNA ligase seals.
Eukaryotic DNA Eukaryotic genome has multiple origins of replication. Replication begins at a
polymerases consensus sequence of AT base pairs.
Eukaryotes have separate polymerases (, , , , ) for synthesizing RNA primers,
leading-strand DNA, lagging-strand DNA, mitochondrial DNA, and DNA repair.
DNA repair: single Single-strand, excision repairspecic glycosylase recognizes and removes damaged
strand base. Endonuclease makes a break several bases to the 5 side. Exonuclease removes
short stretch of nucleotides. DNA polymerase lls gap. DNA ligase seals.
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3'
5'
3'
5'
5'
3'
Leading strand
Primase
Okazaki
fragment
DNA
ligase
Lagging
strand
RNA
primer
DNA
polymerase III
Single-strand
binding protein
DNA
polymerase III
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DNA repair defects Xeroderma pigmentosum (skin sensitivity to UV light), ataxia-telangiectasia (x-rays),
Blooms syndrome (radiation), and Fanconis anemia (cross-linking agents).
Xeroderma Defective excision repair such as uvr ABC Autosomal recessive.
pigmentosum endonuclease. Results in inability to repair
thymidine dimers, which form in DNA when
exposed to UV light.
Associated with dry skin and with melanoma and
other cancers.
DNA/RNA/protein DNA and RNA are both synthesized 5 3. Imagine the incoming
synthesis direction Remember that the 5 of the incoming nucleotide nucleotide bringing a gift
bears the triphosphate (energy source for bond). (triphosphate) to the 3 host.
The 3 hydroxyl of the nascent chain is the target. BYOP (phosphate) from 5
Protein synthesis also proceeds in the 5 to 3 to 3.
direction. Amino acids are linked N
to C.
Types of RNA mRNA is the largest type of RNA. Massive, Rampant, Tiny.
rRNA is the most abundant type of RNA.
tRNA is the smallest type of RNA.
RNA polymerases
Eukaryotes RNA polymerase I makes rRNA. I, II, and III are numbered as
RNA polymerase II makes mRNA. their products are used in
RNA polymerase III makes tRNA. protein synthesis.
No proofreading function, but can initiate chains. -amanitin is found in death
RNA polymerase II opens DNA at promoter site cap mushrooms.
(A-T-rich upstream sequenceTATA and CAAT).
-amanitin inhibits RNA polymerase II.
Prokaryotes RNA polymerase makes all 3 kinds of RNA.
Start and stop AUG (or rarely GUG) is the mRNA initiation codon. AUG inAUGurates
codons EukaryotesAUG codes for methionine, which protein synthesis.
may be removed before translation is completed.
Prokaryotesthe initial AUG codes for a formyl-
methionine (f-met).
Stop codonsUGA, UAA, UAG. UGA = U Go Away.
UAA = U Are Away.
UAG = U Are Gone.
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Thymidine
dimer
3'
5'
5'
3'
UV
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BI OCHEMI STRYMOLECULAR ( cont i nued)
Regulation of gene expression
Promoter Site where RNA polymerase and multiple other Promoter mutation commonly
transcription factors bind to DNA upstream from results in dramatic in
gene locus. amount of gene transcribed.
Enhancer Stretch of DNA that alters gene expression by binding
transcription factors. May be located close to, far
from, or even within (in an intron) the gene whose
expression it regulates.
Operator Site where negative regulators (repressors) bind.
Introns vs. Exons contain the actual genetic information INtrons stay IN the nucleus,
exons coding for protein. whereas EXons EXit and are
Introns are intervening noncoding segments of DNA. EXpressed.
Splicing of mRNA Introns are precisely spliced out of 1 mRNA transcripts. A lariat-shaped intermediate
is formed. Small nuclear ribonucleoprotein particles (snRNP) facilitate splicing by
binding to 1 mRNA transcripts and forming spliceosomes.
RNA processing Occurs in nucleus. After transcription: Only processed RNA is
(eukaryotes) 1. Capping on 5 end (7-methyl-G) transported out of the
2. Polyadenylation on 3 end ( 200 As) nucleus.
3. Splicing out of introns
Initial transcript is called heterogeneous nuclear
RNA (hnRNA).
Capped and tailed transcript is called mRNA.
tRNA structure 7590 nucleotides, cloverleaf form, anticodon end is opposite 3 aminoacyl end. All
tRNAs, both eukaryotic and prokaryotic, have CCA at 3 end along with a high
percentage of chemically modied bases. The amino acid is covalently bound to the 3
end of the tRNA.
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DNA
mRNA
Exons
Introns
Transcription
and splicing
HO-AAAA
3'
5'
Cap
G
pppp
Tail
Coding
mRNA
Methionine
Codon
Anticodon
(CAU) UAC
AUG
ACC
3'
3'
5'
5'
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tRNA charging Aminoacyl-tRNA synthetase (1 per aa, uses ATP) Aminoacyl-tRNA synthetase
scrutinizes aa before and after it binds to tRNA. If and binding of charged
incorrect, bond is hydrolyzed by synthetase. The tRNA to the codon are
aa-tRNA bond has energy for formation of peptide responsible for accuracy of
bond. A mischarged tRNA reads usual codon but amino acid selection.
inserts wrong amino acid.
tRNA wobble Accurate base pairing is required only in the rst 2 nucleotide positions of an mRNA
codon, so codons differing in the 3rd wobble position may code for the same
tRNA/amino acid.
Protein synthesis Met sits in the P sitepeptidyl. The incoming ATPtRNA Activation
amino acid binds to the A siteaminoacyl, (charging).
hydrolyzing Mets bond to its tRNA while GTPtRNA Gripping and
simultaneously forming a peptidyl bond between Going places (translocation).
the 2 amino acids. The ribosome shifts 1 codon
toward the 3 end of the mRNA, shifting the
uncharged tRNA into the E position and the
dipeptidyl tRNA into the P site.
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AA
ATP AMP + PP
i
OH
3'
5'
Aminoacyl tRNA
synthetase
3' 5'
Ribosome
40S
E P A
60S
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BI OCHEMI STRYCELLULAR
Enzyme kinetics
The lower the K
m
, the higher
the afnity.
HINT: Competitive inhibitors
cross each other
competitively, while
noncompetitive inhibitors
do not.
Enzyme regulation Enzyme concentration alteration (synthesis and/or destruction), covalent modication
methods (e.g., phosphorylation), proteolytic modication (zymogen), allosteric regulation (e.g.,
feedback inhibition), and transcriptional regulation (e.g., steroid hormones).
Cell cycle phases M (mitosis: prophasemetaphase G stands for Gap or Growth; S
anaphasetelophase) for Synthesis.
G
1
(growth)
S (synthesis of DNA)
G
2
(growth)
G
0
(quiescent G
1
phase)
G
1
and G
0
are of variable duration. Mitosis is
usually shortest phase. Most cells are in G
0
.
Rapidly dividing cells have a shorter G
1
.
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V
max
V
max
K
m
[S]
Noncompetitive inhibitor
Uninhibited
Competitive inhibitor
slope =
1
K
m
K
m
V
max
1
V
max
1
[S]
1
[S]
1
V
1
V
V
e
l
o
c
i
t
y
(
V
)
1
2
K
m
= [S] at V
max
1
2
Competitive Noncompetitive
inhibitors inhibitors
Resemble substrate Yes No
Overcome by [S] Yes No
Bind active site Yes No
Effect on V
max
Unchanged
Effect on K
m
Unchanged
G
2
Mitosis
G
1
S
phase
Interphase
(G
1
, S, G
2
)
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Rough RER is the site of synthesis of secretory (exported) Mucus-secreting goblet cells of
endoplasmic proteins and of N-linked oligosaccharide addition the small intestine and
reticulum (RER) to many proteins. antibody-secreting plasma
cells are rich in RER.
Nissl bodies Nissl bodies (in neurons)rough ER; not found in axon or axon hillock.
Synthesize enzymes (e.g., ChAT) and peptide neurotransmitters.
Smooth SER is the site of steroid synthesis and detoxication Liver hepatocytes and
endoplasmic of drugs and poisons. steroid hormoneproducing
reticulum (SER) cells of the adrenal cortex
are rich in SER.
Functions of Golgi 1. Distribution center of proteins and lipids from I-cell disease is caused by the
apparatus ER to the plasma membrane, lysosomes, and failure of addition of
secretory vesicles mannose-6-phosphate to
2. Modies N-oligosaccharides on asparagine lysosome proteins, causing
3. Adds O-oligosaccharides to serine and threonine these enzymes to be secreted
residues outside the cell instead of
4. Proteoglycan assembly from proteoglycan core being targeted to the
proteins lysosome. Characterized by
5. Sulfation of sugars in proteoglycans and of coarse facial features and
selected tyrosine on proteins restricted joint movement.
6. Addition of mannose-6-phosphate to specic
lysosomal proteins, which targets the protein
to the lysosome
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RER
SER
Cell
membrane
Cell
membrane
trans
face
cis
face
(Reproduced, with permission, from Junqueira L, Carneiro J. Basic Histology, 10th ed.
New York:McGraw-Hill, 2003.)
9610_02.2-Biochem 11/9/05 7:24 PM Page 83
BI OCHEMI STRYCELLULAR ( cont i nued)
Microtubule Cylindrical structure 24 nm in diameter and of variable Drugs that act on microtubules:
length. A helical array of polymerized dimers of - 1. Mebendazole/thiabendazole
and -tubulin (13 per circumference). Each dimer (antihelminthic)
has 2 GTP bound. Incorporated into agella, cilia, 2. Taxol (antibreast cancer)
mitotic spindles. Grows slowly, collapses quickly. 3. Griseofulvin (antifungal)
Microtubules are also involved in slow axoplasmic 4. Vincristine/vinblastine
transport in neurons. (anti-cancer)
5. Colchicine (anti-gout)
Chdiak-Higashi syndrome
is due to a microtubule
polymerization defect
resulting in phagocytosis.
Cilia structure 9 + 2 arrangement of microtubules. Kartageners syndrome is due
Dynein is an ATPase that links peripheral to a dynein arm defect,
9 doublets and causes bending of cilium by resulting in immotile cilia.
differential sliding of doublets. Dynein = retrograde.
Kinesin = anterograde.
Plasma membrane Plasma membranes contain cholesterol ( 50%, promotes membrane stability),
composition phospholipids ( 50%), sphingolipids, glycolipids, and proteins. High cholesterol or
long saturated fatty acid content melting temperature. Only noncytoplasmic side
of membrane contains glycosylated lipids or proteins (i.e., the plasma membrane is an
asymmetric, uid bilayer).
Phosphatidylcholine Phosphatidylcholine (lecithin) is a major component of RBC membranes, of myelin, of
function bile, and of surfactant (DPPCdipalmitoyl phosphatidylcholine). Also used in
esterication of cholesterol (LCAT is lecithin-cholesterol acyltransferase).
Sodium pump Na
+
-K
+
ATPase is located in the plasma membrane Ouabain inhibits by binding to
with ATP site on cytoplasmic side. For each ATP K
+
site. Cardiac glycosides
consumed, 3 Na
+
go out and 2 K
+
come in. (digoxin, digitoxin) also
During cycle, pump is phosphorylated. inhibit the Na
+
-K
+
ATPase,
causing cardiac contractility.
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24 nm
Microtubule
doublets
Dynein ATPase
Cytosolic
side
3Na
+
ATP
ADP
3Na
+
2K
+
2K
+
Extracellular
side
P
P
9610_02.2-Biochem 11/9/05 7:24 PM Page 84
G-protein-linked 2nd messengers
Receptor G-protein class
HAVe 1 M&M.
MAD 2s.
Collagen types Collagen is the most abundant protein in the Be Cool, Read Books.
human body. Functions to organize and
strengthen extracellular matrix.
Type I (90%)Bone, tendon, skin, dentin, fascia, Type I: BONE.
cornea, late wound repair.
Type IICartilage (including hyaline), vitreous Type II: carTWOlage.
body, nucleus pulposus.
Type III (Reticulin)skin, blood vessels, uterus,
fetal tissue, granulation tissue.
Type IVBasement membrane or basal lamina. Type IV: Under the oor
Type Xepiphyseal plate. (basement membrane).
Collagen synthesis Inside broblasts:
and structure 1. Collagen chains (preprocollagen)
translated on RERusually Gly-X-Y
polypeptide (X and Y are proline,
hydroxyproline, or hydroxylysine)
2. ER hydroxylation of specic proline
and lysine residues (requires vitamin C)
3. Golgi glycosylation of pro--chain
lysine residues and formation of
procollagen (triple helix of 3 collagen
chains)
4. Procollagen molecules are exocytosed
into extracellular space
Outside broblasts:
5. Procollagen peptidases cleave terminal
regions of procollagen, transforming
procollagen into insoluble tropocollagen
6. Many staggered tropocollagen molecules
are reinforced by covalent lysine-hydroxylysine
cross-linkage (by lysyl oxidase) to make collagen
brils
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Receptor
Lipids
PIP
2
G
q H
1
,
1
, V
1,
M
1
, M
3
IP
3 [Ca
2+
]
in
DAG Protein
kinase C
Phospholipase C
G
s
1
,
2
, D
1
,
H
2
, V
2
Protein kinase A
ATP
cAMP
Receptor
Adenylyl cyclase
M
2
,
2
, D
2
G
i
Receptor
+
mRNA
Nucleus
Glycosylation
(pro chain)
Triple helix (procollagen)
Osteogenesis
imperfecta
Collagen fibrils
with crosslinks
OH OH
OH OH
ER
DNA
Golgi
Ehlers-Danlos
Scurvy
Hydroxylation
Cell membrane
Peptide cleavage
c(1-)
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BI OCHEMI STRYCELLULAR ( cont i nued)
Ehlers-Danlos Faulty collagen synthesis causing:
syndrome 1. Hyperextensible skin
2. Tendency to bleed (easy bruising)
3. Hypermobile joints
10 types. Inheritance varies. Associated with berry aneurysms.
Osteogenesis Primarily an autosomal-dominant disorder caused May be confused with child
imperfecta by a variety of gene defects, resulting in abnormal abuse.
collagen synthesis. Clinically characterized by: Type II is fatal in utero and in
1. Multiple fractures occurring with minimal the neonatal period.
trauma (brittle bone disease), which may Incidence is 1:10,000.
occur during the birth process
2. Blue sclerae due to the translucency of the
connective tissue over the choroid
3. Hearing loss (abnormal middle ear bones)
4. Dental imperfections due to lack of dentition
Immunohistochemical stains
Stain Cell type
Vimentin Connective tissue
Desmin Muscle
Cytokeratin Epithelial cells
Glial brillary acid proteins (GFAP) Neuroglia
Neurolaments Neurons
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BI OCHEMI STRYMETABOLI SM
Metabolism sites
Mitochondria Fatty acid oxidation (-oxidation), acetyl-CoA production, Krebs cycle.
Cytoplasm Glycolysis, fatty acid synthesis, HMP shunt, protein synthesis (RER), steroid synthesis
(SER).
Both Gluconeogenesis, urea cycle, heme synthesis.
Summary of pathways
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Glycogen
UDP-glucose Glucose-1-phosphate
Glucose
Glucose-6-phosphate 6-phosphogluconolactone
Fructose-6-phosphate
Fructose-1,6-bisphosphate
Glyceraldehyde-3-P DHAP
1,3-bis-phosphoglycerate
3-phosphoglycerate
2-phosphoglycerate
Phosphoenolpyruvate (PEP)
Pyruvate
Acetyl-CoA
Glyceraldehyde
Ribulose-5-phosphate
F1P Fructose
NH
4
+ CO
2
Carbamoyl
phosphate
Citrulline
Aspartate
Argininosuccinate
Urea
cycle
Ornithine
Urea
H
2
O
Arginine
Fumarate
Oxaloacetate
Malate
TCA
cycle
Succinate
Citrate
Isocitrate
-ketoglutarate
Succinyl-CoA Methylmalonyl-CoA
Propionyl-CoA
Odd-chain
fatty acids
Acetoacetate -hydroxybutyrate
Mevalonate
Galactose
Galactose-1-phosphate
HMP shunt
Glycolysis
Lactate
Acetoacetyl-CoA HMG-CoA
Malonyl-CoA Fatty acids
Cholesterol
1
2
3 4
5
6
7 8
9
11
12
Gluconeogenesis
15
14
16
17
18
10
1 Galactokinase (mild galactosemia)
2 Galactose-1-phosphate uridyltransferase
(severe galactosemia)
3 Hexokinase/glucokinase
4 Glucose-6-phosphatase (von Gierkes)
5 Glucose-6-phosphate dehydrogenase (G6PD)
6 Transketolase
7 Phosphofructokinase
8 Fructose-1,6-bisphosphatase
9 Fructokinase (essential fructosuria)
10 Aldolase B (fructose intolerance)
11 Pyruvate kinase
12 Pyruvate dehydrogenase
13 HMG-CoA reductase
14 Pyruvate carboxylase
15 PEP carboxykinase
16 Citrate synthase
17 -ketoglutarate dehydrogenase
18 Ornithine transcarbamylase
13
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BI OCHEMI STRYMETABOLI SM ( cont i nued)
ATP Base (adenine), ribose, 3 phosphoryls. 2 phosphoanhydride bonds, 7 kcal/mol each.
Aerobic metabolism of glucose produces 38 ATP via malate shuttle, 36 ATP via G3P shuttle.
Anaerobic glycolysis produces only 2 net ATP per glucose molecule.
ATP hydrolysis can be coupled to energetically unfavorable reactions.
Activated carriers Phosphoryl (ATP).
Electrons (NADH, NADPH, FADH
2
).
Acyl (coenzyme A, lipoamide).
CO
2
(biotin).
1-carbon units (tetrahydrofolates).
CH
3
groups (SAM).
Aldehydes (TPP).
Glucose (UDP-glucose).
Choline (CDP-choline).
S-adenosyl- ATP + methionine SAM. SAM transfers methyl SAMthe methyl donor man.
methionine units to a wide variety of acceptors (e.g., in
synthesis of phosphocreatine, a high-energy
phosphate active in muscle ATP production).
Regeneration of methionine (and thus SAM) is
dependent on vitamin B
12
.
Signal molecule ATP cAMP via adenylate cyclase.
precursors GTP cGMP via guanylate cyclase.
Glutamate GABA via glutamate decarboxylase (requires vitamin B
6
).
Choline ACh via choline acetyltransferase (ChAT).
Arachidonate prostaglandins, thromboxanes, leukotrienes via cyclooxygenase/
lipoxygenase.
Fructose-6-P fructose-1,6-bis-P via phosphofructokinase (PFK), the rate-limiting
enzyme of glycolysis.
1,3-BPG 2,3-BPG via bisphosphoglycerate mutase.
Universal electron Nicotinamides (NAD
+
, NADP
+
) and avin NADPH is a product of the
acceptors nucleotides (FAD
+
). HMP shunt.
NAD
+
is generally used in catabolic processes to
carry reducing equivalents away as NADH.
NADPH is used in anabolic processes (steroid NADPH is used in:
and fatty acid synthesis) as a supply of reducing 1. Anabolic processes
equivalents. 2. Respiratory burst
3. P-450
B
I
O
C
H
E
M
I
S
T
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Y
H
I
G
H
-
Y
I
E
L
D
P
R
I
N
C
I
P
L
E
S
NH
2
OOO
-
O P P P
H O H O
N
NN
N
O
O O O
O
-
O
-
O
-
~ ~
9610_02.2-Biochem 11/9/05 7:24 PM Page 88
89
Oxygen-dependent respiratory burst
Hexokinase vs. Hexokinase is found throughout body. Only hexokinase is feedback
glucokinase GLucokinase is primarily found in the Liver inhibited by G6P.
(lower afnity [ K
m
] but higher capacity Glucokinase phosphorylates
[ V
max
]). excess glucose (e.g., after a
meal) to sequester it in the
liver as G6P.
Glycolysis D-glucose Glucose-6-phosphate Glucose-6-P .
regulation,
Hexokinase/glucokinase
*
irreversible Fructose-6-P Fructose-1,6-BP ATP , AMP , citrate ,
enzymes
Phosphofructokinase-1
fructose-2,6-BP .
(rate-limiting step)
Phosphoenolpyruvate Pyruvate ATP , alanine ,
Pyruvate kinase
fructose-1,6-BP .
Pyruvate Acetyl-CoA ATP , NADH ,
Pyruvate
acetyl-CoA .
dehydrogenase
* Glucokinase in liver; hexokinase in all other tissues.
Glycolytic enzyme Hexokinase, glucose phosphate isomerase, aldolase, RBCs metabolize glucose
deciency triosephosphate isomerase, phosphate glycerate anaerobically (no
kinase, enolase, and pyruvate kinase deciencies mitochondria) and thus
are associated with hemolytic anemia. depend solely on glycolysis.
H
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Phagolysosome
Neutrophil
cell membrane
O
2
O
2
H
2
O
2
HOCl
NADPH
NADP
+
H
2
O
2
H
2
O
GSH GSSG
NADP
+
NADPH
G6P 6PG
Cl
-
GSH/GSSG = glutathione
(reduced/oxidized)
HOCl
= bleach
Bacteria
1 NADPH oxidase (deficiency =
chronic granulomatous disease)
2 Superoxide dismutase
3 Myeloperoxidase
4 Catalase
5 Glutathione reductase
6 Glucose-6-phosphate
dehydrogenase (G6PD)
1
2
3
4
5
6
9610_02.2-Biochem 11/9/05 7:24 PM Page 89
BI OCHEMI STRYMETABOLI SM ( cont i nued)
Pyruvate The complex contains 3 enzymes that require 5 The complex is similar to the
dehydrogenase cofactors (the rst 4 B vitamins plus lipoic acid): -ketoglutarate
complex 1. Pyrophosphate (B
1
, thiamine; TPP) dehydrogenase complex
2. FAD (B
2
, riboavin) (same cofactors, similar
3. NAD (B
3
, niacin) substrate and action).
4. CoA (B
5
, pantothenate)
5. Lipoic acid
Reaction: pyruvate + NAD
+
+ CoA acetyl-CoA +
CO
2
+ NADH.
Activated by exercise:
NAD
+
/NADH ratio
ADP
Ca
2+
Pyruvate Causes backup of substrate (pyruvate and alanine), Lysine and Leucinethe only
dehydrogenase resulting in lactic acidosis. Can be seen in purely ketogenic amino
deciency alcoholics due to B
1
deciency. acids.
Findings: neurologic defects.
Treatment: intake of ketogenic nutrients (e.g., high
fat content or lysine and leucine).
Pyruvate metabolism 6 ATP equivalents are needed
to generate glucose from
pyruvate.
Alanine serves as carrier of
amino groups from muscle to
liver.
Oxaloacetate can be used to
replenish TCA cycle or in
gluconeogenesis.
Cori cycle Transfers excess reducing
equivalents from RBCs and
muscle to liver, allowing
muscle to function
anaerobically (net 2 ATP).
90
B
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S
MUSCLE LIVER
B
L
O
O
D
Glucose
2ATP
Pyruvate
Lactate
dehydrogenase
Lactate
Pyruvate
Lactate
Lactate
dehydrogenase
6ATP
Glucose
Lactate
Glucose
Cytosol
Mitochondria
Alanine
NADH + H
+
NAD
+
NADH + H
+
Acetyl-CoA Oxaloacetate
NAD
+
CO
2
+ ATP
CO
2
Pyruvate
ALT LDH
PC
PDH
9610_02.2-Biochem 11/9/05 7:24 PM Page 90
91
TCA cycle Produces 3 NADH, 1 FADH
2
,
2 CO
2
, 1 GTP per acetyl-
CoA = 12 ATP/acetyl-CoA
(2 everything per glucose)
-ketoglutarate dehydrogenase
complex requires same
cofactors as the pyruvate
dehydrogenase complex.
Can I Keep Selling Sex
For Money, Ofcer?
Electron transport chain and oxidative phosphorylation
Electron transport 1 NADH 3 ATP; 1 FADH
2
2 ATP.
chain
Oxidative 1. Electron transport inhibitors (rotenone, antimycin A, CN
, H
+
, CO
2
, 2,3-BPG, and temperature favor When youre Relaxed, you do
regulation T form over R form (shifts dissociation curve to your job better (carry O
2
).
right, leading to O
2
unloading). Fetal hemoglobin (2 and 2
subunits) has lower afnity
for 2,3-BPG than adult
hemoglobin (HbA) and thus
has higher afnity for O
2
.
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2
1
1
2
Heme
Succinyl CoA + Glycine
-Aminolevolinic acid
(ALA)
Porphobilinogen
Pre-uroporphyrinogen
Committed step
ALA synthetase
Lead
poisoning
Heme
Protoporphyrin
Coproporphyrinogen
Uroporphyrinogen III
Porphyria
cutanea tarda
Fe
2
Acute intermittent
porphyria
()
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104
BI OCHEMI STRYMETABOLI SM ( cont i nued)
Methemo- Iron in hemoglobin is in a reduced state (ferrous, Administer nitrites in cyanide
globinemia Fe
2+
). Methemoglobin is an oxidized form of poisoning to oxidize
hemoglobin (ferric, Fe
3+
) that does not bind O
2
hemoglobin to
as readily but has afnity for CN
. methemoglobin form.
Treat toxic levels of
METHemoglobin with
METHylene blue.
CO
2
transport in CO
2
binds to amino acids in globin chain (at N CO
2
must be transported from
blood terminus) but not to heme. CO
2
binding favors T tissue to lungs, the reverse
(taut) form of hemoglobin (and thus promotes O
2
of O
2
(occurs primarily in the
unloading). form of bicarbonate).
B
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O
C
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9610_02.2-Biochem 11/9/05 7:24 PM Page 104
Specific IgG
in patients
blood
Peroxidase
enzyme
generates
color
Specific
antigen in
patients blood
Test antibody
1.
2.
Test
antigen
105
BI OCHEMI STRYLABORATORY TECHNI QUES
Polymerase chain Molecular biology laboratory procedure that is used to synthesize many copies of a desired
reaction (PCR) fragment of DNA.
Steps:
1. DNA is denatured by heating to generate 2 separate strands
2. During cooling, excess premade DNA primers anneal to a specic sequence on each
strand to be amplied
3. Heat-stable DNA polymerase replicates the DNA sequence following each primer
These steps are repeated multiple times for DNA sequence amplication.
Molecular biology techniques
Southern blot A DNA sample is electrophoresed on a gel and then SNoW DRoP:
transferred to a lter. The lter is then soaked in a Southern = DNA
denaturant and subsequently exposed to a labeled Northern = RNA
DNA probe that recognizes and anneals to its Western = Protein
complementary strand. The resulting double-
stranded labeled piece of DNA is visualized
when the lter is exposed to lm.
Northern blot Similar technique, except that Northern blotting
involves radioactive DNA probe binding to
sample RNA.
Western blot Sample protein is separated via gel electrophoresis
and transferred to a lter. Labeled antibody is
used to bind to relevant protein.
Enzyme-linked A rapid immunologic technique testing for ELISA is used in many
immunosorbent antigen-antibody reactivity. laboratories to determine
assay (ELISA) Patients blood sample is probed with either whether a particular
1. Test antigen (coupled to color-generating antibody (e.g., anti-HIV) is
enzyme)to see if immune system present in a patients blood
recognizes it; or sample. Both the sensitivity
2. Test antibody (coupled to color-generating and the specicity of ELISA
enzyme)to see if a certain antigen is approach 100%, but both
present false positive and false
If the target substance is present in the sample, negative results do occur.
the test solution will have an intense color
reaction, indicating a positive test result.
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BI OCHEMI STRYGENETI CS
Genetic terms
Variable expression Nature and severity of the phenotype varies from 1 individual to another.
Incomplete Not all individuals with a mutant genotype show the mutant phenotype.
penetrance
Pleiotropy 1 gene has > 1 effect on an individuals phenotype.
Imprinting Differences in phenotype depend on whether the mutation is of maternal or paternal
origin (e.g., AngelMans syndrome [Maternal], Prader-Willi syndrome [Paternal]).
Anticipation Severity of disease worsens or age of onset of disease is earlier in succeeding generations
(e.g., Huntingtons disease).
Loss of heterozygosity If a patient inherits or develops a mutation in a tumor suppressor gene, the complementary
allele must be deleted/mutated before cancer develops. This is not true of oncogenes.
Dominant negative Exerts a dominant effect. A heterozygote produces a nonfunctional altered protein that
mutation also prevents the normal gene product from functioning.
Linkage Tendency for certain alleles at 2 linked loci to occur together more often than
disequilibrium expected by chance. Measured in a population, not in a family, and often varies in
different populations.
Mosaicism Occurs when cells in the body have different genetic makeup (e.g., lyonization
random X inactivation in females).
Locus heterogeneity Mutations at different loci can produce the same phenotype (e.g., albinism).
Hardy-Weinberg If a population is in Hardy-Weinberg equilibrium, Hardy-Weinberg law assumes:
population then: 1. There is no mutation
genetics Disease prevalence: p
2
+ 2pq + q
2
= 1 occurring at the locus
Allele prevalence: p + q = 1 2. There is no selection for
p and q are separate alleles; 2pq = heterozygote any of the genotypes at
prevalence. the locus
3. Mating is completely
random
4. There is no migration into or
out of the population
being considered
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Modes of inheritance
Autosomal dominant Often due to defects in structural genes. Many Often pleiotropic and, in many
generations, both male and female, affected. cases, present clinically after
puberty. Family history
crucial to diagnosis.
Autosomal recessive 25% of offspring from 2 carrier parents are affected. Commonly more severe than
Often due to enzyme deciencies. Usually seen in dominant disorders; patients
only 1 generation. often present in childhood.
X-linked recessive Sons of heterozygous mothers have a 50% chance of Commonly more severe in
being affected. No male-to-male transmission. males. Heterozygous females
may be affected.
X-linked dominant Transmitted through both parents. Either male or Hypophosphatemic rickets.
female offspring of the affected mother may
be affected, while all female offspring of the
affected father are diseased.
Mitochondrial Transmitted only through mother. All offspring of Lebers hereditary optic
inheritance affected females may show signs of disease. neuropathy; mitochondrial
myopathies.
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carrier
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BI OCHEMI STRYGENETI CS ( cont i nued)
Autosomal-dominant diseases
Adult polycystic kidney Always bilateral, massive enlargement of kidneys due to multiple large cysts. Patients
disease present with pain, hematuria, hypertension, progressive renal failure. 90% of cases
are due to mutation in APKD1 (chromosome 16). Associated with polycystic liver
disease, berry aneurysms, mitral valve prolapse. Juvenile form is recessive.
Familial Elevated LDL owing to defective or absent LDL receptor. Heterozygotes (1:500) have
hypercholesterolemia cholesterol 300 mg/dL. Homozygotes (very rare) have cholesterol 700+ mg/dL,
(hyperlipidemia severe atherosclerotic disease early in life, and tendon xanthomas (classically in the
type IIA) Achilles tendon); MI may develop before age 20.
Marfans syndrome Fibrillin gene mutation connective tissue disorders.
Skeletal abnormalitiestall with long extremities (arachnodactyly), hyperextensive
joints, and long, tapering ngers and toes (see Image 109).
Cardiovascularcystic medial necrosis of aorta aortic incompetence and
dissecting aortic aneurysms. Floppy mitral valve.
Ocularsubluxation of lenses.
Neurobromatosis Findings: caf-au-lait spots, neural tumors, Lisch nodules (pigmented iris
type 1 (von hamartomas). Also marked by skeletal disorders (e.g., scoliosis),
Recklinghausens pheochromocytoma, and tumor susceptibility. On long arm of chromosome
disease) 17; 17 letters in von Recklinghausen.
Neurobromatosis Bilateral acoustic neuroma, optic pathway gliomas, juvenile cataracts. NF2 gene on
type 2 chromosome 22; type 2 = 22.
Tuberous sclerosis Findings: facial lesions (adenoma sebaceum), hypopigmented ash leaf spots on skin,
cortical and retinal hamartomas, seizures, mental retardation, renal cysts, cardiac
rhabdomyomas. Incomplete penetrance, variable presentation.
Von HippelLindau Findings: hemangioblastomas of retina/cerebellum/medulla; about half of affected
disease individuals develop multiple bilateral renal cell carcinomas and other tumors.
Associated with deletion of VHL gene (tumor suppressor) on chromosome 3 (3p).
Von HippelLindau = 3 words for chromosome 3.
Huntingtons disease Findings: depression, progressive dementia, choreiform movements, caudate atrophy,
and levels of GABA and ACh in the brain. Symptoms manifest in affected
individuals between the ages of 20 and 50. Gene located on chromosome 4; triplet
repeat disorder. Hunting 4 food.
Familial adenomatous Colon becomes covered with adenomatous polyps after puberty. Progresses to colon
polyposis cancer unless resected. Deletion on chromosome 5; 5 letters in polyp.
Hereditary Spheroid erythrocytes; hemolytic anemia; increased MCHC. Splenectomy is curative.
spherocytosis
Achondroplasia Autosomal-dominant cell-signaling defect of broblast growth factor (FGF) receptor 3.
Results in dwarsm; short limbs, but head and trunk are normal size.
Autosomal- Cystic brosis, albinism,
1
-antitrypsin deciency, phenylketonuria, thalassemias,
recessive sickle cell anemias, glycogen storage diseases, mucopolysaccharidoses (except Hunters),
diseases sphingolipidoses (except Fabrys), infant polycystic kidney disease, hemochromatosis.
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109
Cystic brosis Autosomal-recessive defect in CFTR gene on Infertility in males due to absent
chromosome 7. Defective Cl