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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