1. Enzymatic Catalysis III Ribonuclease A An example of a general acid and base catalysisAn 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 catalystDigestive enzyme found in pancreas - involved in digestion of RNA. (ribonucleic acid)Both a general acid and general base catalyst RNA not DNARNA not DNA Cleaves between the 5' P of one sugar residue and the 2' O of the other riboseCleaves between the 5' P of one sugar residue and the 2' O of the other ribose Pyrimidines are only real recognition sitePyrimidines are only real recognition site

2. Binding site of ribonuclease -x-ray crystallography- Use a non-hydrolyzable analog - phosphonateUse a non-hydrolyzable analog - phosphonate Catalytic cleft - larger and more open than lysozymeCatalytic cleft - larger and more open than lysozyme The protein binds by salt bridges with phosphate backboneThe protein binds by salt bridges with phosphate backbone –lysine and arginine Pyrimidine binds in active site purines are too bigPyrimidine binds in active site purines are too big –3 amino acids: His 12, His 119 and Lys41

3. Catalytic mechanism Iodoacetate - alkylates histidinesIodoacetate - 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 How can we find it- transition state very fast – slow down (temp) – slow/no reacting analogs (ATP-  S) Non-hydrolyzable version of ATP Non-hydrolyzable version of ATP NAG 3 - binds and is slow to react NAG 3 - binds and is slow to react competitive inhibitors work well (why) 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 catalytic cleft - hydrogen bonds, ionic bonds and van der Waal contacts occur with substrate in active site – NAG 3 fits part way in site Use modeling to determine rest of sugar polymer position 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: – NAG 3 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 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) 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 leads to increase in pK Glu normal - R-pK = 4.25 Glu normal - R-pK = 4.25 Glu 35 - R-pK ~ 5.0 Glu 35 - R-pK ~ 5.0

14. Transition State - proposed only Glu 35 can donate H+ only Glu 35 can donate H+ donates H+ to glycosidic bond (general acid) donates H+ to glycosidic bond (general acid) leaves sugar ring w/ (+) charge -unstable intermediate leaves sugar ring w/ (+) charge -unstable intermediate promoted by several stabilization factors 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 (+) C 1 reacts with water (H 3 O - ) (+) C 1 reacts with water (H 3 O - ) diffusion of products diffusion of products

15. Transition State - proposed promoted by several stabilization factors 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 (+) C 1 reacts with water (H 3 O - ) (+) C 1 reacts with water (H 3 O - ) diffusion of products diffusion of products

16. Transition State - proposed promoted by several stabilization factors 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 (+) C 1 reacts with water (H 3 O - ) (+) C 1 reacts with water (H 3 O - ) diffusion of products diffusion of products

17. Evidence for proposed transition state mechanism Cleavage pattern Cleavage pattern – earlier A-F pattern based on model – actual NAG 4 and NAG 2 products made Transition state analogs Transition state analogs – change NAG so it is in a permanent 1/2 chair conformation – analog binds 3000 times faster than normal NAG 3 pH vs. catalytic rate 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