Enzymatic Catalysis III

1
Enzymatic Catalysis III

• Ribonuclease A
• An example of a general acid and base catalysis
• Digestive enzyme found in pancreas - involved in
  digestion of RNA. (ribonucleic acid)Both a
  general acid and general base catalyst
• RNA not DNA
• Cleaves between the 5' P of one sugar residue and
  the 2' O of the other ribose
• Pyrimidines are only real recognition site

2

• Binding site of ribonuclease -x-ray
  crystallography-
• Use a non-hydrolyzable analog - phosphonate
• Catalytic cleft - larger and more open than
  lysozyme

• The protein binds by salt bridges with phosphate
  backbone
• lysine and arginine
• Pyrimidine binds in active site purines are too
  big
• 3 amino acids His 12, His 119 and Lys41

3

• Catalytic mechanism
• Iodoacetate - alkylates histidines
• selective iodonation inhibits ribonuclease
  activity
• pH curve is most active at pH 7 - indicates
  histadine involvement

4

• Catalytic mechanism
• This is a hydrolytic reaction yet the reaction
  begins with without water
• The reaction occurs by the following mechanism
• His 12 (deprotonated) accepts the H of 2OH

5

• Catalytic mechanism
• The reaction occurs by the following mechanism
• Nucleophilic attack by 2 O on P

6

• Catalytic mechanism
• Simultaneously - His 119 (protonated) donates H
  to other side of phosphate bond.
• Lysine stabilizes (-) of phosphate
• When His 12 and 119 are done cyclic O-P-O is
  formed

7

• Catalytic mechanism
• roles of His119 and 12 are reversed when water
  is added onto 2O and P

8

• Transition State
• Pentacovalent trigonal bipyramid
• The attacking and leaving groups are in line
• The intermediate is stabilized by positive
  charged amino acids

9
Lysozyme

• Structure and background
• Endogenous protection system - lysozyme - attacks
  cell wall
• N -acetylglucosamine NAG N -Acetylmuramate NAM
• Cell wall strengthen by polymers of NAG-NAM
  through glycosidic bonds alpha beta 1 - 4
  linkages
• Lysozyme cleaves beta (1-4) bonds.
• 1st 3D structure known - highly studied first
  discovered by Flemming (he also found penicillin)

10

• Lysozyme
• Small compact enzyme few alpha helix beta
  sheets - 4 disulfide bridges
• Binding site open along one side of protein

11

• Binding site of lysozyme -x-ray crystallography-
• How can we find it- transition state very fast
• slow down (temp)
• slow/no reacting analogs (ATP-? S)
• Non-hydrolyzable version of ATP
• NAG3 - binds and is slow to react
• competitive inhibitors work well (why)
• mutant proteins that bind but not react with
  substrate
• catalytic cleft - hydrogen bonds, ionic bonds and
  van der Waal contacts occur with substrate in
  active site
• NAG3 fits part way in site
• Use modeling to determine rest of sugar polymer
  position
• distortion of D-ring to fit with the rest of the
  sugars
• Strain effect

12

• Which ring of the sugar polymer is cleaved -
  answer determined based on x-ray structure and
  other known facts, such as
• NAG3 little reactivity - not here
• NAM-NAG at 3rd bond wont fit (NAM lactyl chain)
• only D-E site left
• Now which part of the bond
• Heavy water adds only to D ring

13

• Use x-ray structure to find which amino acids are
  involved
• General acid hydrolysis involved in this type
  catalysis
• find a H donator (acidic amino acids)
• look near binding site for culprit aa
• Asp 52 - tied up in polar environment - H bonded
• Glu 35 - in non-polar environment not bonded
• leads to increase in pK
• Glu normal - R-pK 4.25
• Glu 35 - R-pK 5.0

14

• Transition State - proposed
• only Glu 35 can donate H
• donates H to glycosidic bond (general acid)
• leaves sugar ring w/ () charge -unstable
  intermediate
• promoted by several stabilization factors
• charged ring intermediate - carbonium ion
• Asp 52 helps to stabilize for next step to occur
• strain on ring structure also help stabilization
• rearrangement allows for resonance of electrons
• () C1 reacts with water (H3O-)
• diffusion of products

15

• Transition State - proposed
• promoted by several stabilization factors
• charged ring intermediate - carbonium ion
• Asp 52 helps to stabilize for next step to occur
• metal does this for inorganic acid hydrolysis
• strain on ring structure also help stabilization
• rearrangement allows for resonance of electrons
• () C1 reacts with water (H3O-)
• diffusion of products

16

• Transition State - proposed
• promoted by several stabilization factors
• charged ring intermediate - carbonium ion
• Asp 52 helps to stabilize for next step to occur
• metal does this for inorganic acid hydrolysis
• strain on ring structure also help stabilization
• rearrangement allows for resonance of electrons
• () C1 reacts with water (H3O-)
• diffusion of products

17

• Evidence for proposed transition state mechanism
• Cleavage pattern
• earlier A-F pattern based on model
• actual NAG4 and NAG2 products made
• Transition state analogs
• change NAG so it is in a permanent 1/2 chair
  conformation
• analog binds 3000 times faster than normal NAG3
• pH vs. catalytic rate
• activity follows charge state of glutamate
• Modification of amino acids - add ester group on
  Asp 52 leads to inactive enzyme - can not promote
  carbonium ion w/o charge