23 - transpeptidase 2

Question 1

describe the serine protease active site

Answer 1

• there are four key features in the active site
  
  • serine side chain
  • a His and Asp is always close by, Asp at physiological level is in negative form and acts as a base
  • His and Asp work together as a super base and store proton on side of Ser
  • oxyanion hole is made of amide back bone, sometimes on same chain or different chain
  • O- charge is created and is stabilized
  • the three is called catalytic triad, then you have the oxyanion hole
• the production of an amide bond is what the serine protease does, capable of doing it in reverse as well

Question 2

describe the reaction of amide hydrolysis in solution

Answer 2

• At pH 7.4 half life is hundreds of year → amide in water will be hydrolized in water after thousands of years
• the components have to approach from a certain distance and particular alignment to each other, which makes it slow
• hydroxide and hydronium is what is needed
• hydroxide as used as a Nu, causing double bond to break
• orbitals of one functional group have to line up with the other functional groups exactly. bc OH exist little, the chance that it carries out perfectly is small which is why its slow
• needs enough momentum too
• the tetrahedral intermediate has negative charge, not stable, it CAN reverse
• trajectory, distance and momentum
• O can grab H from water and becomes neutral intermediate, difficult to do, can be reversed again
• to complete hydrolysis, amine group needs to be modified to become a better leaving group- by protonation
• double bond then deprotonated by water in last sequence, can happen easily, but first three are unlikely and not easy to do

Question 3

describe the reaction of amide hydrolysis in enzyme

Answer 3

• first of all enzyme interacts with enzyme with non bonding interactions, like H bond stuff that fit perfectly the substrate
• on the enzyme the serine side chain is OH, is going to act like special molecule like OH and is the special Nu
• the histidine and asp are ready made base, perfect angle and distance from serine
• base removes proton from OH, instead of electron going to O, it goes as a Nu to the amide and break the double bond above
• now tetrahedral intermediate, it’s negative, this is where the oxyanion hole comes into place. it has H bond donors on it though in perfect place to stabilize it- don’t need protonation, it can be stabilized with negative charge
• nitrogen gets protonated with the conjugate base
• acyl enzyme

Question 4

describe the formation of a new amide bond

Answer 4

Formation of cross link between D-Ala and 6-aminopimelic acid
Look at lecture for picture

Question 5

what is the role of water in the formation of new amide bonds

Answer 5

• Hydrolysis of one D-Ala-D-Ala peptide bond
• first step is the same as mechanism as cross link) d ala gets cut off from d ala as over there, leaving group.)
• the second reaction: water as Nu, N changes to O, Asp removes proton, electrons donate to carbonyl which causes negative charge, oxyanion stabilizes, when bond reforms, a dfifferent alignment allowes it to grab the proton. carboyxalte is free and enzyme is regenerated

Question 6

what is the role of the catalytic triad and the oxyanion hole

Answer 6

look at notes

Question 7

describe the opening of the β-lactam by enzyme

Answer 7

• in penicillin it has amide where N is connected at beta carbon
• it acts as a special electrophile, its reactivity is like below
• net result is the acyl enzyme, same as when transpeptidase works on side chain

Question 8

describe the covalent inhibition that occurs between penicillin and transpeptidase

Answer 8

look at notes

Question 9

what does it mean for penicillin to be an irreversable inhibitor

Answer 9

• Inhibition is permanent
  
  • Enzyme unable to hydrolyze the ester formed
• Cell wall formation is prevented
  
  • Cells burst from turgor (internal pressure) when dividing

Question 10

describe the selectivity of penicillin

Answer 10

• No equivalent enzyme- don’t need to worry about penicillin attacking our body
• D-Ala-D-Ala linkage is unique in nature- the substrate is different and unique
  
  • Bacteria only
  • Virtually all other amino acids have L configuration

Question 11

what makes penicillin a clean drug

Answer 11

• Non-toxic
• Very safe
• Few side effects
• Major side effect is allergy (~ 0.1 % incidence)

Question 12

describe the source of the penicillin allergy

Answer 12

• Penicillin is a good electrophile
• Can acylate host serine proteases or other proteins
• Acylate host (human) serine proteases
• “tagged” protein recognized by immune system
• Strongest reactions happen second time taking drug