4-Proteins

Question 1

What is the active site?

Answer 1

• Site where S binds E with weak forces (with charged and polar residues interactions)
• Displacement of water makes hydrophobic interactions important
• Multiple contacts (may be >10)
• Site composed of binding & catalytic residues
• Stabilizes transition state between S and P

Question 2

What is the lock and key model?

Answer 2

1st model of ES binding (1890 Fischer) that proposes that each enzyme has a specific site (the lock) that exactly matches the substrate (the key).

Question 3

What is the induced fit model?

Answer 3

In late 1950s, Koshland proposed that enzymes sometimes had to change their shape to accommodate the chemicals and that this shape could be part of the catalytic reaction.

Question 4

What are some characteristics of the active site?

Answer 4

• Within a subunit, but some between two subunits
• Catalytic residues are usually nucleophiles
• aa chains in catalysis have atypical pKa (His)
• Catalytic residues widely separated in 1ry seq.

Question 5

What is the catalytic action in chymotryspsin?

Answer 5

• It’s a serine protease that cleaves peptide bonds
• Ser residue loses proton & becomes nucleophile
• Catalytic triad: 3 side chains that interact to transfer charge through space (Asp, His, Ser)
  1. Cleavage of peptide bond by activated serine
  2. Release of one half of the polypeptide & retention of half through covalent bond to serine
  3. Covalent bond is cut by a hydrolysis reaction
  4. Release of other half of polypeptide and restoration of HO group on serine

Question 6

How specific are enzymes?

Answer 6

• Often only 1 physiological substrate (or 1 type)
• Most can act as analogs
• Hydrolases and transferases have broader specificities
• Stereospecificity requires multipoint attachment

Question 7

How specific are trypsin and thrombin?

Answer 7

1. Trypsin cleaves peptide bond after a Lys or Arg

2. Thrombin cleaves peptide bonds between Arg and Glycine.

Question 8

What is a way different proteases have such different specificity?

Answer 8

Same broad function and similar structures, BUT the S pockets have distinct residues that create a micro-environment that determines specificity.

1. Chymotrypsin has a neutral pocket
2. Trypsin has a negatively charged pocket (Asp)
3. Elastase has valine side chains, so hydrophobic groups bind best.

Question 9

What are the types of reversible inhibition?

Answer 9

1. Competitive: inhibitor competes directly with binding of S to the enzyme
2. Non-competitive: acts at different site on enzyme from the S binding site; indifferent to S

Question 10

What happens to Lineweaver-Burk plot with a competitive inhibitor?

Answer 10

• Competitive inhibitor does NOT change Vmax
• Km value increases because more S is needed to overcome the presence of inhibitor
• Slope increases because 1/Km is more negative and 1/V remains the same

Question 11

What is Pravastatin?

Answer 11

It is a common competitive inhibitor used to treat high cholesterol. It binds to HMG-CoA-reductase active site.

Question 12

What happens to Lineweaver-Burk plot with a non-competitive inhibitor?

Answer 12

• Non-competitive inhibitor decreases Vmax
• Km value remains the same
• The effect cannot be overcome adding more substrate, but the amount of S needed to reach Vmax in each case is the same

Question 13

What are some examples of common irreversible enzyme inhibitors?

Answer 13

• Penicillin: covalently binds transpeptidase; preventing synthesis of bacterial cell wall
• Aspirin:m odifies active site serine by acetylation in COX-1/2 preventing inflammation

Question 14

What are irreversible inhibitors?

Answer 14

• Cannot be overcome with high [S]
• Overcome only by new protein synthesis
• Usually a result of covalent modification of the enzyme.

Question 15

What are allosteric effectors?

Answer 15

• Molecules that regulate enzymes through binding at allosteric sites.
• Can decrease or increase the affinity of an enzyme for substrate.
• Can change the maximum catalytic activity
• Can change the specificity of the enzyme

Question 16

What is ribonucleotide reductase? how is it regulated?

Answer 16

• Enzyme that catalyzes the converison of ribo-nt diphosphates to deoxyribonucleotides disphosphates to make dNTPs.
• Has 1 active site and 2 allosteric sites
  a) Activity allosteric site: increase with ATP & decrease activity with dATP binding
  b) Specificity allosteric site: dGTP binding increases specificity for ADP as substrate

Question 17

What are possible structural requirements of enzyme catalysis?

Answer 17

• Correctly folded polypeptide & oligomeric assembly
• Co/post-translational modifications
• Co-factors (generally act like substrates)
• Prosthetic groups (usually covalently attached)
• Metal ions

Question 18

What are some characteristics of metal cofactors?

Answer 18

• widely distributed
• some are trace elements
• most are common elements (Zn, Mn, Ca)
• can be directly involved in catalysis or play a structural role

Question 19

What is the role of vitamins in enzymes?

Answer 19

• They can act as enzyme cofactors

- Involved in hydroxylation of lysine and proline, which is important in collagen formation

Question 20

What are the major types of collagen?

Answer 20

A. Fibril forming 
   I. skin, bone, tendon, cornea, BVs
   II. cartilage, vertebral disks, vitreous body
   III. BVs, fetal skin
B. Network-forming
   IV. basement membrane
   VII. beneath stratified squamous epithelia
C. Fibril-associated
   IX. cartilage 
   XII. tendon, ligaments

Question 21

What is collagen’s 1ry sequence?

Answer 21

Lots of repeats of: -Gly-X-Y

• X is often proline
• Y is often hydroxylysine

Question 22

What is scurvy?

Answer 22

-Disease caused by vitamin C deficiency, which causes impaired assembly of collagen helices and fibers because of the failure to hydroxylate prolines and lysines. Fibers that are formed have less tensile strength than normal.

Question 23

What is required to hydroxylate prolines?

Answer 23

Prolyl hydroxylase, Fe2+, ascorbate (vitaminC), O2

Question 24

What are the steps in collagen synthesis?

Answer 24

1. Genes transcribed into mRNAs for pro-a1/a2
2. mRNA translated in ER and extruded into ER lumen where signal sequence is removed
3. Some Pro and Lys residues are hydroxylated
4. Someh-Lys residues are glycosyated
5. Three pro-a chains assemble; intra & inter chain S-S bonds form at C-terminal
6. a triple helix is formed; procollagen is produced
7. Procallogen is secreted from Golgi into ECM
8. N & C-terminal propeptides are cleaved by peptidases (for later crosslinking between fibers) producing tropocollagen
9. Self-assembly of tropocollagen into fibrils
10. Cross-linking to form mature collagen fibers

Question 25

What is Osteogenesis imperfecta type II?

Answer 25

• Disease caused by a replacement of a Glycine residue by a bulky aa in the collagen chain.
• The severity of the disease is increased if the mutation is near the C-terminus.
• No tightening of wrapping of collagen at the ends
• Bone malformations