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Structure and Mechanism II: Ribonuclease A Frazer Li.

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1 Structure and Mechanism II: Ribonuclease A Frazer Li

2 Outline Introduction –Some history of RNase A –As well as the Function and Structure Folding and Stability –How RNase A binds to RNA The Reaction Mechanism Reaction Energetics and Homologues Conclusion

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 A RNase A is the first enzyme and third protein to have a correct amino acid sequence Was first crystallized over 50 years ago.

4 History A variety of methods were used to determine the structure of RNase A 1) 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 Function 2 Classes of enzyme that catalyze the synthesis or degradation of RNA: 1) RNA polymerase – synthesis 2) RNA depolymerases or “ribonucleases” – degradation RNase A has been the object of landmark work on the folding stability and chemistry of proteins in enzymology and in molecular evolution RNA essential for life!

6 Structure The size of RNase A is small -Has 124 amino acid residues -Contains 19 of the 20 natural amino acids, lacking only tryptophan Has similar shape to a kidney with active site residues lying in the cleft

7 Structure Long four-stranded antiparallel β-sheet and three short α-helixes -Cross-linked by four disulfide bonds involving all eight of its cysteine residues -peptide 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 enzyme – –more stability from Cys26-Cys84 and Cys58-Cys110 than from Cys65-Cys72 and Cys40-Cys95 -two proline residues with cis peptide bonds The stability of RNase A is legendary The two proline residues with cis peptide bonds, and the three residues most important for catalysis is His12, His119, and Lys41

9 RNA Binding SUBSITES B1, B2, and B3 interact with the bases of a bound substrate The 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 base B3 has a preference for a purine base

10 RNA Binding SUBSITES Three other enzymic subsites (P0, P1, and P2) interact with the phosphoryl groups of a bound substrate The 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 residue ONE-DIMENSIONAL DIFFUSION The abilitiy to diffuse in one dimension can accelerate the formation of a site-specific interaction within a linear biopolymer by up to fold. Such facilitated diffusion is used by transcription factors and restriction endonucleases to locate specific sites on double-stranded DNA Specifically, 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 solvent For 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 atom This attack displace a nucleoside His119 acts as an acid that protonates the 5’’-oxygen to facilitate its displacement Both products are released to solvent The side chain of Lys41 and the main chain of Phe120 enhance catalysis by stabilizing this transition state

14 Reaction Mechanism RNase 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 119 Only one histidine residue is alkylated in each molecule of RNase A. The rate of the single enzymic carboxymethylation is nearly fold greater than that of free histidine The alkylation, which causes a marked decrease in catalytic activity, modifies only His12 or His119 Catalysis by RNase A has a classic bell-shaped pH rate profile This profile is consistent with a mechanism that involves two titratable residues, one protonated and the other unprotonated

16 Important Residues for Catalysis His12 and His119 Eliminating the imidazole group of His12 decreases the affinity of the enzyme for this transition state by fold during cleavage of poly(C), UpA, and UpOC 6 H 4 -p-NO 2 Eliminating the imidazole group of His 119 decreased this affinity by 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 groups –pKa of CH 3 OCH 2 CH 2 OH is 14.8 –pKa of UpOC 6 H 4 -p-NO 2 is 7.14 –Thus, the contribution of His119 to catalysis decreases when the pKa of the conjugate acid of the leaving group decreases His119 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 Lys41 contributes to catalytic activity –When Lys41 is replaced by an arginine residue, the variant have approximately 2% of the activity of the wild-type enzyme in hydrolysis Catalytic role of Lys41 is to stabilize the excess negative charge that accumulates on the nonbridging phosphoryl oxygens in transition state during RNA cleavage –Stabilized by Coulombic interactions –By short, strong hydrogen bond involving the partial transfer of a proton from Lys41 Lys41 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 hydrolysis 2’,3’-cyclic phosphodiester intermediate and hydrolysis of this cyclic intermediate to form a 3’-phosphomonoester NMR spectroscopy was used to monitor how this cyclic intermediate accumulates during catalysis by RNase A and small molecules The cyclic intermidiate does not accumulate during catalysis by hydroxide ion or imidazole buffer In the presence of these small-molecule catalysts, hydrolysis of the cyclic intermediate is faster than transphosphorylation of RNA These 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 reaction The imidazole group of His 119 has complementary role, acting as an acid in the trasphosphorylation reaction and a base in the hydrolysis reaction After catalysis of transphosphorylation, each histidine residue in the active site of RNase A is protonated appropriately to catalyze hydrolysis of the bound cyclic intermediate RNase 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 A Enhances Catalysis Similarly, replacing His12 or His119 the base and acid in catalysis slows catalysis by 10 4 to 10 5 fold B2 subsite provides a fold rate acceleration

21 Homologues Humans contain at least five homologues of RNase A –RNase 1 which is from human pancrease –RNase 4 which is from human liver are distinct enzymes –Angiogenin is a plasma enzyme that promotes neovasculariztion –Eosinopholic leukocytes contain RNase 2, which is neurotoxic –RNase 3 which has helinthotoxic and antibacterial activities

22 Conclusion RNase A has been the most studied enzyme of the 20 th century Used to digest RNA produced by stomach microorganisms Methods now exist to produce unlimited quantities of RNase A and it’s homologues Can be used to exploit further use of RNase A in biotechnology and medicine RNase A will continue to be used as a model system


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