Enzymatic Catalysis III

Ribonuclease A 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. Digestive enzyme found in pancreas - involved in digestion of RNA. (ribonucleic acid)Both a general acid and general base catalyst(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 Cleaves between the 5' P of one sugar residue and the 2' O of the other riboseribose

• Pyrimidines are only real recognition sitePyrimidines are only real recognition site

Binding site of ribonuclease -x-ray crystallography-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 The protein binds by salt bridges with salt bridges with phosphate backbonephosphate backbone–lysine and argininelysine and arginine•Pyrimidine binds in Pyrimidine binds in active site purines are active site purines are too bigtoo big–3 amino acids: His 3 amino acids: His 12, His 119 and Lys4112, His 119 and Lys41

Catalytic mechanismCatalytic mechanism• Iodoacetate - alkylates histidines Iodoacetate - alkylates histidines

– selective iodonation inhibits ribonuclease activityselective iodonation inhibits ribonuclease activity– pH curve is most active at pH 7 - indicates histadine pH curve is most active at pH 7 - indicates histadine

involvementinvolvement

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

acidsacids

LysozymeStructure and backgroundStructure and background

Endogenous protection system - lysozyme - Endogenous protection system - lysozyme - attacks cell wallattacks cell wall

N N -acetylglucosamine NAG &-acetylglucosamine NAG &NN -Acetylmuramate -Acetylmuramate NAMNAM

Cell wall strengthen by polymers of NAG-NAM Cell wall strengthen by polymers of NAG-NAM through glycosidic bonds alpha & beta 1 - 4 through glycosidic bonds alpha & beta 1 - 4 linkageslinkages

Lysozyme cleaves beta (1-4) bonds.Lysozyme cleaves beta (1-4) bonds. 1st 3D structure known - highly studied first 1st 3D structure known - highly studied first

discovered by Flemming (he also found discovered by Flemming (he also found penicillin)penicillin)

LysozymeLysozyme Small compact enzyme few alpha helix Small compact enzyme few alpha helix

& beta sheets - 4 disulfide bridges& beta sheets - 4 disulfide bridges Binding site open along one side of Binding site open along one side of

proteinprotein

Binding site of lysozymeBinding site of lysozyme -x-ray crystallography- -x-ray crystallography-• How can we find it- transition state very fastHow can we find it- transition state very fast

– slow down (temp)slow down (temp)– slow/no reacting analogs (ATP-slow/no reacting analogs (ATP- S) S)•Non-hydrolyzable version of ATPNon-hydrolyzable version of ATP

•NAGNAG33 - binds and is slow to react - binds and is slow to react

• competitive inhibitors work well (why)competitive inhibitors work well (why)– mutant proteins that bind but not react with mutant proteins that bind but not react with

substratesubstrate• catalytic cleft - hydrogen bonds, ionic bonds and van catalytic cleft - hydrogen bonds, ionic bonds and van

der Waal contacts occur with substrate in active site der Waal contacts occur with substrate in active site

– NAGNAG33 fits part way in site fits part way in site

• Use modeling to determine rest of sugar polymer Use modeling to determine rest of sugar polymer positionposition

– distortion of D-ring to fit with the rest of the distortion of D-ring to fit with the rest of the sugarssugars

– Strain effectStrain 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 Use x-ray structure to find which amino acids are involvedacids are involved– General acid hydrolysis involved in this General acid hydrolysis involved in this

type catalysistype catalysis•find a H+ donator (acidic amino acids)find a H+ donator (acidic amino acids)– look near binding site for culprit aalook near binding site for culprit aa– Asp 52 - tied up in polar environment - H Asp 52 - tied up in polar environment - H

bondedbonded– Glu 35 - in non-polar environment not Glu 35 - in non-polar environment not

bondedbonded• leads to increase in pK leads to increase in pK •Glu normal - R-pK = 4.25Glu normal - R-pK = 4.25•Glu 35 - R-pK ~ 5.0Glu 35 - R-pK ~ 5.0

Transition State - proposedTransition State - proposed• only Glu 35 can donate H+only Glu 35 can donate H+• donates H+ to glycosidic bond donates H+ to glycosidic bond

(general acid)(general acid)• leaves sugar ring w/ (+) charge leaves sugar ring w/ (+) charge

-unstable intermediate -unstable intermediate • promoted by several promoted by several

stabilization factorsstabilization factors– charged ring intermediate - charged ring intermediate -

carbonium ioncarbonium ion– Asp 52 helps to stabilize for Asp 52 helps to stabilize for

next step to occurnext step to occur– strain on ring structure also strain on ring structure also

help stabilization help stabilization – rearrangement allows for rearrangement allows for

resonance of electronsresonance of electrons

• (+) C(+) C11 reacts with water (H reacts with water (H33OO--))

• diffusion of productsdiffusion of products

Transition State - proposedTransition State - proposed• promoted by several stabilization factorspromoted by several stabilization factors

– charged ring intermediate - carbonium ioncharged ring intermediate - carbonium ion– Asp 52 helps to stabilize for next step to occurAsp 52 helps to stabilize for next step to occur– metal does this for inorganic acid hydrolysismetal does this for inorganic acid hydrolysis– strain on ring structure also help stabilization strain on ring structure also help stabilization – rearrangement allows for resonance of electronsrearrangement allows for resonance of electrons

• (+) C(+) C11 reacts with water (H reacts with water (H33OO--))

• diffusion of productsdiffusion of products

Transition State - proposedTransition State - proposed• promoted by several stabilization factorspromoted by several stabilization factors

– charged ring intermediate - carbonium ioncharged ring intermediate - carbonium ion– Asp 52 helps to stabilize for next step to occurAsp 52 helps to stabilize for next step to occur– metal does this for inorganic acid hydrolysismetal does this for inorganic acid hydrolysis– strain on ring structure also help stabilization strain on ring structure also help stabilization – rearrangement allows for resonance of electronsrearrangement allows for resonance of electrons

• (+) C(+) C11 reacts with water (H reacts with water (H33OO--))

• diffusion of productsdiffusion of products

Evidence for proposed transition state mechanismEvidence for proposed transition state mechanism• Cleavage pattern Cleavage pattern

– earlier A-F pattern based on modelearlier A-F pattern based on model

– actual NAGactual NAG44 and NAG and NAG22 products made products made

• Transition state analogsTransition state analogs– change NAG so it is in a permanent 1/2 chair change NAG so it is in a permanent 1/2 chair

conformationconformation

– analog binds 3000 times faster than normal NAGanalog binds 3000 times faster than normal NAG33

• pH vs. catalytic ratepH vs. catalytic rate– activity follows charge state of glutamateactivity follows charge state of glutamate

Modification of amino acids - add ester group on Asp 52 Modification of amino acids - add ester group on Asp 52 leads to inactive enzyme - can not promote carbonium ion leads to inactive enzyme - can not promote carbonium ion w/o + chargew/o + charge