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 2’OH

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 2’O 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