Structure and Mechanism II:Structure and Mechanism II:Ribonuclease ARibonuclease A

Frazer LiFrazer Li

OutlineOutline

IntroductionIntroduction– Some history of RNase ASome history of RNase A– As well as the Function and StructureAs well as the Function and Structure

Folding and Stability Folding and Stability – How RNase A binds to RNAHow RNase A binds to RNA

The Reaction MechanismThe Reaction Mechanism

Reaction Energetics and HomologuesReaction Energetics and Homologues

ConclusionConclusion

HistoryHistory

Ribonucleolytic activity in the pancreas of ruminants is Ribonucleolytic activity in the pancreas of ruminants is highhigh– possibly to digest large amount of RNA produced by stomach possibly to digest large amount of RNA produced by stomach

microorganisms. microorganisms.

This high level of activity has led to the discovery of This high level of activity has led to the discovery of Ribonuclease ARibonuclease A

RNase A is the first enzyme and third protein to have a RNase A is the first enzyme and third protein to have a correct amino acid sequencecorrect amino acid sequence

Was first crystallized over 50 years ago.Was first crystallized over 50 years ago.

HistoryHistory

A variety of methods were used to determine the structure A variety of methods were used to determine the structure of RNase Aof RNase A

1) Fast atom bombardment mass spectrometry 1) Fast atom bombardment mass spectrometry (FABMS) - assigns disulfide bonds of (FABMS) - assigns disulfide bonds of

a protein with RNase A.a protein with RNase A.2) Work on RNase A has yielded the first 3D 2) Work on RNase A has yielded the first 3D

structure of a protein containing an structure of a protein containing an isoaspartyl residue, derived isoaspartyl residue, derived

from from deamidation of an deamidation of an asparagine residue (Asn 67) asparagine residue (Asn 67)

3) NMR spectroscopy in elaborating both protein 3) NMR spectroscopy in elaborating both protein structure and protein folding pathways.structure and protein folding pathways.

FunctionFunction

2 Classes of enzyme that catalyze the synthesis or 2 Classes of enzyme that catalyze the synthesis or degradation of RNA:degradation of RNA:

1) RNA polymerase – synthesis1) RNA polymerase – synthesis

2) RNA depolymerases or “ribonucleases” – degradation2) RNA depolymerases or “ribonucleases” – degradation

RNase A has been the object of landmark work on the RNase A has been the object of landmark work on the folding stability and chemistry of proteins in enzymology folding stability and chemistry of proteins in enzymology and in molecular evolutionand in molecular evolution

RNA essential for life!RNA essential for life!

StructureStructure

The size of RNase A is smallThe size of RNase A is small- Has 124 amino acid residuesHas 124 amino acid residues- Contains 19 of the 20 natural amino acids, lacking only tryptophanContains 19 of the 20 natural amino acids, lacking only tryptophan

Has similar shape to a kidney with active site residues lying in the Has similar shape to a kidney with active site residues lying in the cleftcleft

StructureStructure

Long four-stranded antiparallel Long four-stranded antiparallel ββ-sheet and three short -sheet and three short αα-helixes-helixes- Cross-linked by four disulfide bonds involving all eight of its Cross-linked by four disulfide bonds involving all eight of its

cysteine residuescysteine residues- peptide bonds preceding two of the four proline residues are in peptide bonds preceding two of the four proline residues are in

the the ciscis conformation conformation

Folding and StabilityFolding 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

RNA BindingRNA BindingSUBSITESSUBSITES

B1, B2, B1, B2, andand B3 B3 interact with interact with the bases of a bound substratethe bases of a bound substrateThe The B1B1 subsite bind only subsite bind only pyrimidine bases pyrimidine bases (demonstrates an (demonstrates an approximately 30-fold kinetic approximately 30-fold kinetic preference for cytosine-preference for cytosine-containing versus uracil-containing versus uracil-containing substrates)containing substrates)The The B2B2 and and B3B3 subsites bind subsites bind all bases, but all bases, but B2B2 has a has a preference for an adenine preference for an adenine base base B3B3 has a preference for a has a preference for a purine basepurine base

RNA BindingRNA Binding

SUBSITESSUBSITES

Three other enzymic subsites Three other enzymic subsites ((P0P0, , P1P1, and , and P2P2) interact with ) interact with the phosphoryl groups of a the phosphoryl groups of a bound substratebound substrateThe enzyme catalyzes the The enzyme catalyzes the cleavage of the P-O bond of a cleavage of the P-O bond of a phosphoryl group bound in the phosphoryl group bound in the P1 subsite, which is the active P1 subsite, which is the active sitesite

RNA BindingRNA Binding

SUBSTRATE SPECIFICITYSUBSTRATE SPECIFICITYRNase A catalyzes the cleavage of the P-O bond of an RNA strand 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 and the hydrolysis of the P-O bond of a nucleoside 2’,3’-cyclic phosphodiester on the 3’-side of a pyrimidine residuephosphodiester on the 3’-side of a pyrimidine residue

ONE-DIMENSIONAL DIFFUSIONONE-DIMENSIONAL DIFFUSIONThe abilitiy to diffuse in one dimension can accelerate the formation The abilitiy to diffuse in one dimension can accelerate the formation of a site-specific interaction within a linear biopolymer by up to 10of a site-specific interaction within a linear biopolymer by up to 1033--fold.fold.Such facilitated diffusion is used by transcription factors and Such facilitated diffusion is used by transcription factors and restriction endonucleases to locate specific sites on double-stranded restriction endonucleases to locate specific sites on double-stranded DNADNASpecifically, a uridine nucleotide is cleaved more quickly by RNase 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 A if it is flanked by a long stretch of poly(dA) than if it is flanked by a short stretchshort stretch

RNA BindingRNA Binding

PROCESSIVE CATALYSISPROCESSIVE CATALYSISIn contrast, “processive” enzymes bind a polymeric substrate and In contrast, “processive” enzymes bind a polymeric substrate and catalyze a series of identical chemical reactions along that polymer catalyze a series of identical chemical reactions along that polymer before releasing it to solventbefore releasing it to solventFor a substrate to be acted on processively, it must contain a For a substrate to be acted on processively, it must contain a repeating structural motifrepeating structural motif

Reaction MechanismReaction MechanismIT IS A GENERAL ACID-BASE IT IS A GENERAL ACID-BASE

CATALYSISCATALYSIS

The side chain of His12 acts as a The side chain of His12 acts as a base that abstracts a proton from base that abstracts a proton from the 2’-oxygen of a substrate the 2’-oxygen of a substrate molecule, and thereby facilitates molecule, and thereby facilitates its attack on the phosphorus its attack on the phosphorus atomatomThis attack displace a nucleosideThis attack displace a nucleosideHis119 acts as an acid that His119 acts as an acid that protonates the 5’’-oxygen to protonates the 5’’-oxygen to facilitate its displacementfacilitate its displacementBoth products are released to Both products are released to solventsolventThe side chain of Lys41 and the The side chain of Lys41 and the main chain of Phe120 enhance main chain of Phe120 enhance catalysis by stabilizing this catalysis by stabilizing this transition statetransition state

Reaction MechanismReaction Mechanism

RNase A catalyze hydrolysis of RNA by a two-step process with the RNase A catalyze hydrolysis of RNA by a two-step process with the intermediate formation of a 2’,3’-cyclic nucleotideintermediate formation of a 2’,3’-cyclic nucleotide

Important Residues for CatalysisImportant Residues for Catalysis

His12 and His 119His12 and His 119Only one histidine residue is alkylated in each molecule of RNase A.Only one histidine residue is alkylated in each molecule of RNase A.

The rate of the single enzymic carboxymethylation is nearly 10The rate of the single enzymic carboxymethylation is nearly 1044-fold -fold greater than that of free histidinegreater than that of free histidine

The alkylation, which causes a marked decrease in catalytic activity, The alkylation, which causes a marked decrease in catalytic activity, modifies only His12 or His119modifies only His12 or His119

Catalysis by RNase A has a classic bell-shaped pH rate profileCatalysis by RNase A has a classic bell-shaped pH rate profile

This profile is consistent with a mechanism that involves two This profile is consistent with a mechanism that involves two titratable residues, one protonated and the other unprotonatedtitratable residues, one protonated and the other unprotonated

Important Residues for CatalysisImportant Residues for Catalysis

His12 and His119His12 and His119Eliminating the imidazole group of His12 decreases the affinity of the Eliminating the imidazole group of His12 decreases the affinity of the enzyme for this transition state by 10enzyme for this transition state by 1044-fold during cleavage of poly(C), UpA, -fold during cleavage of poly(C), UpA, and UpOCand UpOC66HH44-p-NO-p-NO22

Eliminating the imidazole group of His 119 decreased this affinity by 10Eliminating the imidazole group of His 119 decreased this affinity by 1044-fold -fold during cleavage of UpA.during cleavage of UpA.

Therefore, the value of the imidazole group of His 119 to catalysis depends Therefore, the value of the imidazole group of His 119 to catalysis depends on the pKa of the conjugate acid of the leaving groupson the pKa of the conjugate acid of the leaving groups– pKa of CHpKa of CH33OCHOCH22CHCH22OH is 14.8OH is 14.8– pKa of UpOCpKa of UpOC66HH44-p-NO-p-NO22 is 7.14 is 7.14– Thus, the contribution of His119 to catalysis decreases when the pKa of the Thus, the contribution of His119 to catalysis decreases when the pKa of the

conjugate acid of the leaving group decreasesconjugate acid of the leaving group decreases

His119 is proposed to both protonate a nonbridging oxygen of the His119 is proposed to both protonate a nonbridging oxygen of the phosphate anion and deprotonate this same oxygen in the phosphorane phosphate anion and deprotonate this same oxygen in the phosphorane intermediate intermediate

Important Residues for CatalysisImportant Residues for CatalysisLys41Lys41

Lys41 contributes to catalytic Lys41 contributes to catalytic activityactivity– When Lys41 is replaced by an When Lys41 is replaced by an

arginine residue, the variant have arginine residue, the variant have approximately 2% of the activity of approximately 2% of the activity of the wild-type enzyme in hydrolysisthe wild-type enzyme in hydrolysis

Catalytic role of Lys41 is to Catalytic role of Lys41 is to stabilize the excess negative stabilize the excess negative charge that accumulates on the charge that accumulates on the nonbridging phosphoryl oxygens nonbridging phosphoryl oxygens in transition state during RNA in transition state during RNA cleavage cleavage – Stabilized by Coulombic Stabilized by Coulombic

interactionsinteractions– By short, strong hydrogen bond By short, strong hydrogen bond

involving the partial transfer of a involving the partial transfer of a proton from Lys41proton from Lys41

Lys41 is also used to donate a Lys41 is also used to donate a single hydrogen bond to the single hydrogen bond to the transition state during catalysistransition state during catalysis

Reaction EnergeticsReaction EnergeticsRNase catalyzes Exergonic reactionsRNase catalyzes Exergonic reactions

Catalyzes both the reverse of transphosphorylation and hydrolysisCatalyzes both the reverse of transphosphorylation and hydrolysis

2’,3’-cyclic phosphodiester intermediate and hydrolysis of this cyclic 2’,3’-cyclic phosphodiester intermediate and hydrolysis of this cyclic intermediate to form a 3’-phosphomonoesterintermediate to form a 3’-phosphomonoester

NMR spectroscopy was used to monitor how this cyclic intermediate NMR spectroscopy was used to monitor how this cyclic intermediate accumulates during catalysis by RNase A and small moleculesaccumulates during catalysis by RNase A and small molecules

The cyclic intermidiate does not accumulate during catalysis by hydroxide ion The cyclic intermidiate does not accumulate during catalysis by hydroxide ion or imidazole bufferor imidazole buffer

In the presence of these small-molecule catalysts, hydrolysis of the cyclic In the presence of these small-molecule catalysts, hydrolysis of the cyclic intermediate is faster than transphosphorylation of RNAintermediate is faster than transphosphorylation of RNA

These results suggest that RNase A has evolved primarily to catalyze These results suggest that RNase A has evolved primarily to catalyze transphosphorylation rather than hydrolysistransphosphorylation rather than hydrolysis

Reaction EnergeticsReaction EnergeticsTherefore, RNase A is referred to RNA depolymeraseTherefore, RNase A is referred to RNA depolymerase

The imidazole group of His12 acts as a base in the transphosphorylation The imidazole group of His12 acts as a base in the transphosphorylation reaction and an acid in the hydrolysis reactionreaction and an acid in the hydrolysis reaction

The imidazole group of His 119 has complementary role, acting as an acid The imidazole group of His 119 has complementary role, acting as an acid in the trasphosphorylation reaction and a base in the hydrolysis reactionin the trasphosphorylation reaction and a base in the hydrolysis reaction

After catalysis of transphosphorylation, each histidine residue in the active After catalysis of transphosphorylation, each histidine residue in the active site of RNase A is protonated appropriately to catalyze hydrolysis of the site of RNase A is protonated appropriately to catalyze hydrolysis of the bound cyclic intermediatebound cyclic intermediate

RNase A short-curcuits this cycle by releasing rather than hydrolyzing the RNase A short-curcuits this cycle by releasing rather than hydrolyzing the cyclic intermediate. cyclic intermediate.

Thus, RNase A has an iso mechanism in which the protonation states of the 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 unliganded enzyme are interconverted by a pathway that does not involve substrate moleculessubstrate molecules

Reaction EnergeticsReaction Energetics

RATE ENHANCEMENTRATE ENHANCEMENT

Replacing Lys41 with an alanine residue removes a potential Replacing Lys41 with an alanine residue removes a potential hydrogen-bond donor from the active site of RNase Ahydrogen-bond donor from the active site of RNase A

Enhances CatalysisEnhances Catalysis

Similarly, replacing His12 or His119 the base and acid in catalysis Similarly, replacing His12 or His119 the base and acid in catalysis slows catalysis by 10slows catalysis by 1044 to 10 to 1055 fold fold

B2 subsite provides a 10B2 subsite provides a 1044-fold rate acceleration-fold rate acceleration

HomologuesHomologues

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

ConclusionConclusion

RNase A has been the most studied enzyme of the 20RNase A has been the most studied enzyme of the 20 thth century century

Used to digest RNA produced by stomach microorganismsUsed to digest RNA produced by stomach microorganisms

Methods now exist to produce unlimited quantities of RNase A and Methods now exist to produce unlimited quantities of RNase A and it’s homologues it’s homologues

Can be used to exploit further use of RNase A in biotechnology and Can be used to exploit further use of RNase A in biotechnology and medicinemedicine

RNase A will continue to be used as a model systemRNase A will continue to be used as a model system