Presentation on theme: "Structure and Mechanism II: Ribonuclease A"— Presentation transcript:
1 Structure and Mechanism II: Ribonuclease A Frazer Li
2 Outline Introduction Folding and Stability The Reaction Mechanism Some history of RNase AAs well as the Function and StructureFolding and StabilityHow RNase A binds to RNAThe Reaction MechanismReaction Energetics and HomologuesConclusion
3 History Ribonucleolytic activity in the pancreas of ruminants is high possibly to digest large amount of RNA produced by stomach microorganisms.This high level of activity has led to the discovery of Ribonuclease ARNase A is the first enzyme and third protein to have a correct amino acid sequenceWas first crystallized over 50 years ago.
4 HistoryA variety of methods were used to determine the structure of RNase A1) Fast atom bombardment mass spectrometry (FABMS) - assigns disulfide bonds of a protein with RNase A.2) Work on RNase A has yielded the first 3D structure of a protein containing an isoaspartyl residue, derived from deamidation of an asparagine residue (Asn 67)3) NMR spectroscopy in elaborating both protein structure and protein folding pathways.
5 Function2 Classes of enzyme that catalyze the synthesis or degradation of RNA:1) RNA polymerase – synthesis2) RNA depolymerases or “ribonucleases” – degradationRNase A has been the object of landmark work on the folding stability and chemistry of proteins in enzymology and in molecular evolutionRNA essential for life!
6 Structure The size of RNase A is small Has 124 amino acid residuesContains 19 of the 20 natural amino acids, lacking only tryptophanHas similar shape to a kidney with active site residues lying in the cleft
7 StructureLong four-stranded antiparallel β-sheet and three short α-helixesCross-linked by four disulfide bonds involving all eight of its cysteine residuespeptide bonds preceding two of the four proline residues are in the cis conformation
8 Folding and Stability Four disulfide bonds: critical to stability on native enzymemore stability from Cys26-Cys84 and Cys58-Cys110 than from Cys65-Cys72 and Cys40-Cys95 -two proline residues with cis peptide bondsThe stability of RNase A is legendaryThe two proline residues with cis peptide bonds, and the three residues most important for catalysis is His12, His119, and Lys41
9 RNA BindingSUBSITESB1, B2, and B3 interact with the bases of a bound substrateThe B1 subsite bind only pyrimidine bases (demonstrates an approximately 30-fold kinetic preference for cytosine-containing versus uracil-containing substrates)The B2 and B3 subsites bind all bases, but B2 has a preference for an adenine baseB3 has a preference for a purine base
10 RNA BindingSUBSITESThree other enzymic subsites (P0, P1, and P2) interact with the phosphoryl groups of a bound substrateThe enzyme catalyzes the cleavage of the P-O bond of a phosphoryl group bound in the P1 subsite, which is the active site
11 RNA Binding SUBSTRATE SPECIFICITY RNase A catalyzes the cleavage of the P-O bond of an RNA strand and the hydrolysis of the P-O bond of a nucleoside 2’,3’-cyclic phosphodiester on the 3’-side of a pyrimidine residueONE-DIMENSIONAL DIFFUSIONThe abilitiy to diffuse in one dimension can accelerate the formation of a site-specific interaction within a linear biopolymer by up to 103-fold.Such facilitated diffusion is used by transcription factors and restriction endonucleases to locate specific sites on double-stranded DNASpecifically, a uridine nucleotide is cleaved more quickly by RNase A if it is flanked by a long stretch of poly(dA) than if it is flanked by a short stretch
12 RNA Binding PROCESSIVE CATALYSIS In contrast, “processive” enzymes bind a polymeric substrate and catalyze a series of identical chemical reactions along that polymer before releasing it to solventFor a substrate to be acted on processively, it must contain a repeating structural motif
13 Reaction Mechanism IT IS A GENERAL ACID-BASE CATALYSIS The side chain of His12 acts as a base that abstracts a proton from the 2’-oxygen of a substrate molecule, and thereby facilitates its attack on the phosphorus atomThis attack displace a nucleosideHis119 acts as an acid that protonates the 5’’-oxygen to facilitate its displacementBoth products are released to solventThe side chain of Lys41 and the main chain of Phe120 enhance catalysis by stabilizing this transition state
14 Reaction MechanismRNase A catalyze hydrolysis of RNA by a two-step process with the intermediate formation of a 2’,3’-cyclic nucleotide
15 Important Residues for Catalysis His12 and His 119Only one histidine residue is alkylated in each molecule of RNase A.The rate of the single enzymic carboxymethylation is nearly 104-fold greater than that of free histidineThe alkylation, which causes a marked decrease in catalytic activity, modifies only His12 or His119Catalysis by RNase A has a classic bell-shaped pH rate profileThis profile is consistent with a mechanism that involves two titratable residues, one protonated and the other unprotonated
16 Important Residues for Catalysis His12 and His119Eliminating the imidazole group of His12 decreases the affinity of the enzyme for this transition state by 104-fold during cleavage of poly(C), UpA, and UpOC6H4-p-NO2Eliminating the imidazole group of His 119 decreased this affinity by 104-fold during cleavage of UpA.Therefore, the value of the imidazole group of His 119 to catalysis depends on the pKa of the conjugate acid of the leaving groupspKa of CH3OCH2CH2OH is 14.8pKa of UpOC6H4-p-NO2 is 7.14Thus, the contribution of His119 to catalysis decreases when the pKa of the conjugate acid of the leaving group decreasesHis119 is proposed to both protonate a nonbridging oxygen of the phosphate anion and deprotonate this same oxygen in the phosphorane intermediate
17 Important Residues for Catalysis Lys41 contributes to catalytic activityWhen Lys41 is replaced by an arginine residue, the variant have approximately 2% of the activity of the wild-type enzyme in hydrolysisCatalytic role of Lys41 is to stabilize the excess negative charge that accumulates on the nonbridging phosphoryl oxygens in transition state during RNA cleavageStabilized by Coulombic interactionsBy short, strong hydrogen bond involving the partial transfer of a proton from Lys41Lys41 is also used to donate a single hydrogen bond to the transition state during catalysis
18 Reaction Energetics RNase catalyzes Exergonic reactions Catalyzes both the reverse of transphosphorylation and hydrolysis2’,3’-cyclic phosphodiester intermediate and hydrolysis of this cyclic intermediate to form a 3’-phosphomonoesterNMR spectroscopy was used to monitor how this cyclic intermediate accumulates during catalysis by RNase A and small moleculesThe cyclic intermidiate does not accumulate during catalysis by hydroxide ion or imidazole bufferIn the presence of these small-molecule catalysts, hydrolysis of the cyclic intermediate is faster than transphosphorylation of RNAThese results suggest that RNase A has evolved primarily to catalyze transphosphorylation rather than hydrolysis
19 Reaction Energetics Therefore, RNase A is referred to RNA depolymerase The imidazole group of His12 acts as a base in the transphosphorylation reaction and an acid in the hydrolysis reactionThe imidazole group of His 119 has complementary role, acting as an acid in the trasphosphorylation reaction and a base in the hydrolysis reactionAfter catalysis of transphosphorylation, each histidine residue in the active site of RNase A is protonated appropriately to catalyze hydrolysis of the bound cyclic intermediateRNase A short-curcuits this cycle by releasing rather than hydrolyzing the cyclic intermediate.Thus, RNase A has an iso mechanism in which the protonation states of the unliganded enzyme are interconverted by a pathway that does not involve substrate molecules
20 Reaction Energetics RATE ENHANCEMENT Replacing Lys41 with an alanine residue removes a potential hydrogen-bond donor from the active site of RNase AEnhances CatalysisSimilarly, replacing His12 or His119 the base and acid in catalysis slows catalysis by 104 to 105 foldB2 subsite provides a 104-fold rate acceleration
21 Homologues Humans contain at least five homologues of RNase A RNase 1 which is from human pancreaseRNase 4 which is from human liver are distinct enzymesAngiogenin is a plasma enzyme that promotes neovasculariztionEosinopholic leukocytes contain RNase 2, which is neurotoxicRNase 3 which has helinthotoxic and antibacterial activities
22 ConclusionRNase A has been the most studied enzyme of the 20th centuryUsed to digest RNA produced by stomach microorganismsMethods now exist to produce unlimited quantities of RNase A and it’s homologuesCan be used to exploit further use of RNase A in biotechnology and medicineRNase A will continue to be used as a model system