Transcript Catalytic Mechanisms

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
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
Catalytic mechanism
• Iodoacetate - alkylates histidines
– selective iodonation inhibits ribonuclease activity
– pH curve is most active at pH 7 - indicates histadine
involvement
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 2’OH
Catalytic mechanism
The reaction occurs by the following mechanism
– Nucleophilic attack by 2’ O on P
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
Catalytic mechanism
• roles of His119 and 12 are reversed when
water is added onto 2’O and P
Transition State
– Pentacovalent trigonal bipyramid
– The attacking and leaving groups are “in line”
– The intermediate is stabilized by positive charged amino
acids
Structure and background
Lysozyme
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)
Lysozyme
 Small compact enzyme few alpha helix & beta
sheets - 4 disulfide bridges
 Binding site open along one side of protein
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
•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
• 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
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
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
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
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