REVIEW FOR TEST 1

PROTEINS
1. Amino acids side-chain determines character. Amino acids build proteins; the character
of the amino acids determine the character of the protein.
2. Peptide bond planar character - and AA side chains determines folding
3. Structural levels primary to quaternary

interactions between side chains

primary peptide bond
secondary H-bond
tertiary apolar, H-bond, ionic, van der Waals,
S-S bonds (sometimes)
Quaternary non-covalent
(apolar, H-bond, ionic, van der Waals)

folding chaperons, with the use of ATP,

prevent misfolding (to prevent interactions
between hydrophobic side chains)

unfolding denaturation

folding problems Alzheimers disease (amyloidosis),

Kreutzfeld-Jacob diseases (prion disease)
4. Protein composition serum electrophoresis (know normal and examples of disease
patterns; details on albumin)
5. Protein families globular proteins and fibrillar proteins

I. HEMOGLOBIN, MYOGLOBIN
Structure globin protein and heme group (protoporphyrin IX + ferrous ion)
(Please remember interactions between the ring and globin and iron ion and globin)
HB tetramer (cooperativity!); MB monomer
Function HB oxygen transfer; MB oxygen storage
- HB proton, NO and CO2 transfer (do not forget CO2 carried in form of bicarbonate,
too)

Please remember molecular movement during oxygenation (starting from ferrous iron moving to
the plane of the heme)!
Summary of the molecular changes of hemoglobin (Perutz model):
1. Two conformation of hemoglobin exist, the T (tense) in deoxy-Hb and R (relaxed) in
oxy-Hb.
2. T changes to R in the lung where oxygen partial pressure is high.
3. The T-conformation is stabilized by polar interactions, which are broken when Hb binds
oxygen, thus the R-conformation is achieved (Bohr protons are released in the lung).
4. R changes to T in the periphery where oxygen partial pressure is low.
5. The T-conformation can bind 2,3-BPG; 2,3-BPG, thus helps the release of oxygen (Rconformation is changed to T in the periphery).
6. The T-conformation is able to bind protons (Bohr protons bind).
7. Hb exists in all-T or all-R conformation. Thus, when the first subunit of the four is
oxygenated, all will follow, and vice versa (positive cooperativity).
Summary of the effectors influencing oxygen binding to hemoglobin:
1. High pO2 promotes oxygen binding.
2. High pH (alkaline concentration) promotes oxygen binding.
3. High pCO2 weakens oxygen binding.
4. Low pH (high proton concentration) weakens oxygen binding.
5. 2,3-BPG weakens oxygen binding.
6. High temperature weakens oxygen binding.
7. Cooperativity promotes both oxygen binding and release
Hemoglobinopathies: know molecular changes and consequences (unstable Hb, change in
oxygen affinity, methemoglobin, thalassemias both alpha and beta)
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Case report: HbS Glu Val change in beta chain apolar interaction of Hb molecules
precipitation RBC sikles vaso-occlusion
Know symptoms, diagnosis, treatment and biochemical bases for all.

II. IMMUNOGLOBULINS
Structure 2 heavy + 2 light chains
Immunoglobulin fold domains (4 in H, 2 in L)
Function

- antigen binding (N-terminal hypevariable region)

- complement and cell activation (Fc part)
Antibody classes (constant region); antibody specificity (variable region)
Disorder - multiple myeloma (serum electrophoretic pattern)
- autoimmune disorders
- immune deficiencies (serum electrophoretic pattern)

III. FIBRILLAR PROTEINS

1. Intracellular fibrillar proteins
Please remember name, structure and function
Intracellular: Microfilament Actin and myosin
Microtubules alpha and beta tubulins, kinesin and dynein
Intermediate filament keratin (skin and hair)
2. Collagen- fibril forming, fibril associated and network forming
Primary structure
Gly-X-Y triplets where Gly-Pro-Y, Gly-X-Hyp(Hyl) are frequent
Every third AA is Gly
Secondary structure polyproline helix
Tertiary structure triple helix

Hydoxylation of Pro and Lys residues: hydroxylases work with Vitamin C (Scurvy!)
Synthesis of fibril-forming collagens (coll I) see figure, please remember intracellular and
extracellular steps/enzymes

Significance of Allysine
Allysine is an oxidized lysine residue; enzyme is Lysyl oxidase (lysyl amino oxidase)
that requires copper ion for activity.
Allysine side-chains can react with each other or with other Lys residues and form
covalent cross-links between triple helices to stabilize the structure.
It also is important in elastin to form desmosin and isodesmosine crosslinks.

Collagen disorders: how to remember?

See table in lecture (add info from text)
Note: Cutis laxa = occipital horn syndrome = Menkes kinky hair disease = copper
deficiency
Ehlers-Danlos IX = lysyl oxidase deficiency
In general, the disorder can arise from
collagen-related genetic problems (Ehlers-Danlos IV, Osteogenesis imperfecta)
processing enzyme problems (Ehlers-Danlos VI, VII, IX)
cofactor problems (Ehlers-Danlos IX, Scurvy)
3. Elastin coiled structure, crosslinked, lots of hydrophobic amino acids, elastic properties
Elastase to digest, alpha-1 antitrypsin (alpha-1 proteinase inhibitor) to inhibit enzyme see case
report

Alport syndrome
Basement membrane coll IV problem
In kidney basement membrane coll IV 5 chain is affected gene is on X chromosome
males are affected more
If 5 (IV) is not there, the GMB-specific triple helix with 3 (IV) and 4 (IV) cannot be formed
(also found in cochlea and eye), instead 1 (IV) and 2 (IV) chains as well as types V
and VI collagens will be overexpressed.
This affects the structure of the glomerular basement membrane; its filtering function and
kidney function in general. Kidney failure is frequent.
Consequences:
Hematuria and proteinuria (severe)
Cells and proteins can permeate the GBM
Hyperphosphatemia and hypocalcemia (slight, compensated)
In normal kidney: 80-90% of phosphate is re-absorbed from filtrate
In kidney failure the glomerular filtration rate (GFR) is decreased, thus the amount of phosphate
filtered is reduced and retained in plasma which results in decreased levels of S-calcium
because Ca-phosphate is deposited in bone
BUT: decreased levels of S-calcium stimulates PTH secretion which stimulates Ca-release from
bone and stimulates excretion of phosphate via inhibiting tubular re-absorption
Low blood pH:
Normal kidney regenerates bicarbonate ion that maintains pH of the blood
By re-absorption in proximal tubules
By generation in tubular cells from water and carbon dioxide
Tubular cells also generate ammonia from glutamine to promote acid secretion.
In kidney failure, production of bicarbonate and ammonia is reduced.
Acids are retained in serum by decreased GFR.

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ENZYMES
1. Biocatalysts proteins; reduction of activation energy
2. Several enzymes work with coenzymes (vitamins) or cofactors (metal ions)
3. Enzyme kinetics: Michaelis-Menten equation Lineweaver-Burk plot
velocity; initial velocity; Km and Vmax; hyperbolic curve-linearized in LB
4. Serine proteinases (= proteases); complement, digestion, blood clotting
5. Regulation of enzymes
6. Clinical applications enzymes and troponin in MI; use of enzymes in vitro in tests;
urokinase, streptokinase and asparaginase in treatment

I.

II.

Regulation of metabolism
Regulation of pathways
A. feed-back inhibition
B. compartmentalization
Regulation of enzyme activity
A. availability
B. amount
C. activity
C.1. covalent modification
C.1.1. irreversible
C.1.2. reversible
C.2. non-covalent modification
C.2.1. inhibition
C.2.2. allosteric modification

Reminder about C.2.:

Competitive Inhibition:
Competitive inhibitors - structurally similar to the substrate and bind at the substrate -binding
site they will compete with the substrate for binding to the binding site, thus the relative
concentration of the substrate and the inhibitor will determine which one will bind.
Increasing substrate concentrations will displace the non-covalently bound inhibitor by mass
action. Since both of them are there, we need more substrate to reach the half-maximal
velocity Km is increased in the presence of a competitive inhibitor, however, V max will not
change (since it is measured at saturating substrate concentration)
REMEMBER THAT SEVERAL DRUGS ARE COMPETITIVE INHIBITORS (Statins, sulfa
drugs, Methotrexate, COX inhibitors know features and action)

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Figure 5.12. Lippincott Lineweaver-Burk double-reciprocal plot for competitive. A competitive inhibitor binds at
the substrate-binding site and increases Km.

Noncompetitive Inhibition:
A noncompetitive inhibitor binds at the catalytic site, not at the substrate-binding site.
Consequently, the inhibition is not reversed by increasing substrate concentration. Km will
not change; the substrate can still bind to the substrate-binding site with the same affinity
A noncompetitive inhibitor behaves as if it were removing active enzyme consequently
decreased Vmax results.
Km does not change, since the affinity of substrate for enzyme does not change, only the total
amount of free enzyme does.

Figure 5.14. Lippincott Double-reciprocal plot for enzyme subject to reversible noncompetitive inhibition. A
noncompetitive inhibitor binds at site other than the substrate binding site, thus K m does not change, but
apparent Vmax decreases.

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Allosteric enzymes; effectors are of K-type, V-type, positive, negative

Ligands that bind to the allosteric site are called allosteric effectors or modulators and they cause
conformational changes in the enzyme so that the affinity of the enzyme for the substrate
changes.
Positive allosteric effectors increase the enzymes affinity by interaction at the activator site and
negative effectors decrease by interaction at the inhibitory site.
Allosteric enzymes exhibit sigmoidal kinetics (oligomeric proteins)
Allosteric enzymes are of K class or V class.
Effectors alter the K 0.5 (similar to Km), but not Vmax, in K class.
Effectors alter the Vmax, but not K 0.5 in V class.
Negative allosteric effectors move the curve
in K class - toward higher [S] - needs more substrate to reach half max velocity
In V class - toward lower velocity less active enzyme
Positive allosteric effectors move the curve
in K class - toward lower [S] needs lesser substrate to reach half max velocity
In V class - toward higher velocity- more active enzyme

Figure 5. 16 Lippincott: Effects of negative and positive effectors on an allosteric enzyme. A. V class. B. K class.
The substrate concentration that gives half-maximal velocity K 0.5 is altered.

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Serine proteases have similar structure-function relationships

a. Closely related DNA sequences
b. Closely related amino acid sequence, conserved at the active site
c. Similar tertiary structures
d. Substrate specificity of serine proteinases can be different
e. Specific inhibitors developed to regulate the activity of Ser-proteinases (serpins)
f. Common catalytic mechanism
Ser activated by charge-rally system
Synthesized in zymogen form activated by covalent modification
Roles in several processes digestive track, blood coagulation, complement cascade

Hemostasis
Blood clotting (coagulation)
Cellular phase: platelet aggregation, involvement of thromboxanes and prostaglandin I2,
exposure of a new surface, vonWillebrandt factor
Plasma phase: extrinsic (tissue factor) and intrinsic systems (negative charge of new surface),
involvement of blood clotting factors (serine proteinase cascade + tow regulatory proteins),
joining at Factor X
Goal is to form fibrin meshwork (crosslinked by F XIII, which is not a Ser-proteinase).
Role of vitamin K to produce Gla sidechains from Glu in several factors for binding of calcium
and platelet membrane to localize coagulation to the site of damage
Hemophilias (factor VIII or IX deficiency, vonWillebrandt disease)
Anticoagulants: heparin, antithrombin III (inhibitor of serine proteinases), vitamin K antagonists
Proceeded by fibrinolysis the removal of the clot by plasmin (Ser-proteinase that needs to be
activated by urokinase or TPA)

Case report: Alpha-1-antitrypsin deficiency

Mutation may result in intracellular accumulation of the protein cell and tissue destruction
(liver disease); and unavailability of the inhibitory function lung destruction
Mutation results in lack of function - unavailability of the inhibitory function lung destruction
Acquired deficiency destroys inhibitor (smoking) or overloads tissue with enzyme, thus
inhibitor is not enough (silicosis)

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