Structure and Mechanism II: Ribonuclease A

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 103-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 104-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 104-fold during cleavage of poly(C), UpA, and UpOC6H4-p-NO2 • Eliminating 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 groups • pKa of CH3OCH2CH2OH is 14.8 • pKa of UpOC6H4-p-NO2 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 104 to 105 fold • B2 subsite provides a 104-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 20th 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