1. Ribozymes: RNA Enzymes - Function & Role
Pamela Osborn

2. Overview What are ribozymes? Background Structure Future applications
Hairpin, Hammerhead, and RNase P
Future applications
Function & Role of ribozymes within the body
Mechanism: Role of metals in catalysis & nucleobase acid/base catalysis.
Kinetics: Efficiency of ribozymes

3. What Are Ribozymes?
Ribonucleic acid capable of catalyzing a chemical reaction.
Previously thought to be the work of proteins.
Vital for many biological processes
Natural ribozymes catalyze phosphodiester transfer or hydrolysis.
Thought to be remnants of an RNA dominated world.
Could solve the chicken or egg dilemma
“Which comes first, enzymes that do the work of the cell or nucleic acids that carry the information required to produce the enzymes? Nucleic acids as catalysts circumvents this problem”

4. 1989 Nobel Prize in Chemistry: Sidney Altman & Thomas R. Cech
They showed that RNA was capable of splicing itself and joining the genetically important pieces together.
By observing this behavior in unprocessed RNA molecules not in the presence of proteins, they showed that RNA can function as a catalysis.

5. Hairpin Ribozyme Found in plant viruses
Composed of two helical domains containing a small and large internal loop.
These two domains must work together in order to form its active conformation.
“A crystal structure of a hairpin ribozyme bound to an inhibitor RNA has been reported (5). The minor grooves of two irregular helices, stems A and B, dock to form the active site. One of the strands of stem A contains the scissile phosphate (Fig. 1B). In the crystal structure, the nucleotides flanking this phosphate are splayed apart (Fig. 1C), aligning the 2'-OH nucleophile (blocked with a methyl group to prevent cleavage) with the reactive phosphorus and the 5'-oxo leaving group. The in-line conformation (required for the second-order nucleophilic substitution transesterification) and biochemical and biophysical data suggested that the ribozyme-inhibitor cocrystal structure represents the ground state, or precursor, conformation (5)”.

6. “Secondary structure of the catalytic core of the
(ÿ)sTRSV hairpin ribozyme. The four helixes (H1 to H4)
are represented with the two internal loops (A and B).
Postulated base-pairs within loops A and B are depicted
by broken lines (Butcher & Burke, 1994b; Cai & Tinoco,
1996). Wavy arrow represents a UV cross-link observed
between positions U42 and G21 (Butcher & Burke,
1994a). Plain arrow indicates the cleavage site. Note that
residues on the substrate strand are denoted with plus
or minus signs on either side of the cleavage site,
whereas residues on the ribozyme strand are numbered
without sign.”
“The four helical stems that comprise the hairpin ribozyme radiate
from a perfectly base-paired four-way junction. In the crystal
structure, the helices form two coaxial stacks: stem D stacks on A,
and stem C stacks on B. The two stacks cross at around a 608 angle,
and are connected by a pair of anti-parallel crossovers (Figs 1b and
2). The junction allows the central portions of stems A and B to dock
through their minor grooves and form the active site of the
ribozyme. No bound metal ions are observed in the junction. In
the crystal structure of an unrelated four-helix junction formed by a
DNA±RNA hybrid18, a bound metal ion was found in the major
groove of one of the B-form helices. Because the stems of the hairpin
ribozyme are in an A-form conformation near the junction, there is
no equivalent binding site in its structure. Well-ordered metal ions
are observed in the non-canonical moiety of stem B (see below).
These could correspond to the cooperatively bound metal ions
detected by FRET5,7, as tight binding of divalent ions was detected
only in experiments with the intact ribozyme, but not the isolated
fully-base-paired junction19.”

8. Hammerhead Ribozyme Name comes from shape of the secondary structure
“3d structure consists of three helices of variable length and a sequence of 11 unpaired nucleotides surrounding the cleavage site”.

9. “Crystal structure of the initial-state of the hammerhead ribozyme
“Crystal structure of the initial-state of the hammerhead ribozyme. The enzyme strand is shown in red,
the substrate strand in yellow, and the cleavage-site nucleotide, C-17, is highlighted in green. The scissile phosphate
lies between C-17 and A-1.1 as indicated by the arrow. The canonical numbering scheme for the nucleotides and
helices is indicated”.

11. RNase P A ribonuclease that serves as a catalyst
Will often cleave RNA
Catalyzes the maturation of T-RNA

12. “Fig. 1. Illustrations of the secondary structures of different types of RNase P RNA according to Haas and Brown [23]. The left structure represents the paradigm of type A, E. coli RNase P RNA, also termed M1 RNA. The type A M1 RNA is also shown in a schematic form together with type B (Mycoplasma hyopneumoniae), type M (Archaeoglobus fulgidus) and Eukarya (human) RNase P RNAs. The shaded areas represent the P7–P11 region in the specificity domain (here referred to as the TBS region) that interacts with the T-stem-loop region (TSL) of the precursor tRNA (referred to as the TSL/TBS interaction), residue A248 (A248/N−1 interaction), the region in helix P4 suggested to interact with a 2′OH in the acceptor stem of the substrate [128] and the GGU-motif (highlighted in white) that interacts with the 3′ end of the substrate (RCCA–RNase P RNA interaction). Note that the type M (A. fulgidus) and human RNase P RNA lack a GGU-motif (i.e. P15). The solid line in the left structure (between helices P5 and P7) marks the boarder between the specificity and catalytic domains”.

14. Future Applications
Specially engineered ribozyme sheers could be used in
Antiviral medication
Production of plants with antivirus properties
Could potentially be used to correct genetic disorders

15. Multifactoral Catalysis of Ribozymes & Mechanism
SN2 reaction: nucleophilic attack by the 2′-O on the 3′-P, with departure of 5′-O
CLEAVAGE OF RNA:
The chemical mechanism of the cleavage reaction of the nucleolytic ribozymes. The ligation reaction is the reverse of the cleavage reaction.
Three strategies of ribozyme catalysis:
General acid-base catalysis: The hydroxyl group is not a strong nucleophile unless a base removes a proton to create the stronger nucleophile. An acid is needed to facilitate the departure of the base+proton.
Charge stabilization of the transition state of ribozyme cleavage by positively charged (metal) ions promotes cleavage. ((shown in green))
The conformational 3° structure of RNA in certain ribozyme structures (such as hairpin and hammerhead) aligns the 2′-O, 3′-P and 5′-O enzyme-substrate complex for attack (Lilley 2003).
Reaction mechanism: SN2 reaction resulting from nucleophilic attack by the 2′-O on the 3′-P, with departure of 5′-O
Two RNA products: one with a 2′–3′ cyclic phosphate terminus and one with a 5′-OH terminus

16. Ribosome Function: Nucleolytic Cleavage
Transesterification reactions :
Transesterification reactions result in breakage of the backbone
mRNA splicing
Self splicing of introns
Functioning ribozymes are structures that can be found within RNA introns such as Group I (G) and II (A) intron. The hairpin & hammerhead ribozymes (plant viruses), and hepatitis delta virus (human) ribozymes all function this way

17. Ribozymes Function
Nucleotides in RNA can function to catalyze reactions such as peptide bond formation
The peptidyl transferase center of cells (ribosome) is a ribozyme
An important feature of RNA that distinguishes it from DNA is the presence of a hydroxyl group at the 2' position of the ribose sugar. A consequence of the presence of the 2'-hydroxyl group is that in flexible regions of an RNA molecule (that is, not involved in formation of a double helix), it can chemically attack the adjacent phosphodiester bond to cleave or ligate the backbone of other RNA molecules.
Ribozymes cleave only at a specific location, using base-pairing and tertiary interactions to help align the cleavage site within the catalytic functioning core of the ribozyme (Khan, 2005).

18. Ribozymes – pH & Metal Ions
RNA lacks nucleobase functional groups pKa’s – unlikely contributor to ribozyme chemistry. However…
‘Metallozymes’
These reactions are pH dependent. The pH at which these reactions occur depend on the pKa of the nucleobases (3.5 and 4.2 for adenine and cytosine) being perturbed closer to neutral by a highly charged environment. (RNA is highly charged).
Since RNA is charged, metal ions such as Mg2+ or K+ are needed to stabilize folding many secondary and tertiary structures. (Zhi-Jie Tan and Shi-Jie Chen 2008) Also, a metal ion could stabilize the charged transition state electrostatically, possibly by binding to one of the non-bridging oxygen atoms or could form an inner-sphere complex with the RNA, acting as a Lewis acid to assist deprotonation of the attacking nucleophile or stabilization of the charged leaving group (Lilley 2003).
Strobel and Cochrane 2007

19. Acid-Base Catalysis of Nucleobases from RNA
(Adenine)38 functioning as an acid A
H bonds stabilizing transition state
(Guanine) 8 functioning as a base
Bevilacqua, Biochemistry 2003

20. John K. Frederiksena, 1 and Joseph A. Piccirilli 2009
Regions of negative electrostatic potential appear red, while electropositive regions appear blue. The A-rich bulge that coordinates two Mg2+ ions is located in a pocket of highly negative electrostatic potential (“metal ion core”) with values ranging from −80 to −100 kT/e
John K. Frederiksena, 1 and Joseph A. Piccirilli 2009

21. Group I intron splicing promoted by two catalytic metal ions.
The nucleophile (U-1 O3′), scissile phosphate, leaving group (ΩG O3′), and labile bond
Catalytic metal ions
RNA splicing by the group I intron involves two symmetrical phosphoryl transfer reactions (Scott A Strobel and Jesse C Cochran)
The pKa values of the free RNA nucleotides are outside the range considered useful for general acid/base catalysis at physiological pH. The requirement of specific divalent metals for activity suggested that RNA structure serves to position metal ions and substrates in the correct context to perform chemistry (Hanna & Doudna 2000).
Steitz and Steitz proposed a “two-divalent-metal-ion model”: one activates the nucleophile and the other coordinates the leaving group by stabilizing the transition state.
This two-metal ion structure suggests that the group I intron active site is mechanistically equivalent to a large number of protein-based
phosphoryl transferases, including all known DNA and RNA polymerases. (Scott A Strobel and Jesse C Cochran)
Strobel and Cochrane 2007

22. Hairpin Ribozyme Nucleobases interact with ribozyme
Transition state stabilized by hydrogen bonds
Transesterification reaction

23. Transition state stabilized by H-bonds
Hairpin Ribozyme
Transition state stabilized by H-bonds
Transesterification reaction
Ruppert et al, Science 2002

24. Kinetics of Ribozymes Michaelis-Menton Kinetics: E + S ES E + P
Catalytic Efficiency:
Ribozyme Km values comparable to Km values of protein enzymes
Ribozymes KCAT are lower than those values observed for protein enzymes.
KCAT/KM = M-1.min-1
1011 times faster than the un-catalyzed reaction
Ribozymes possess Km values which are comparable to Km values of protein enzymes because all naturally occurring ribozymes catalyze with high substrate specificity (Ryder 2010). The catalytic rate constant describes how efficiently a catalyst converts substrate into product. The values of this constant for ribozymes are markedly lower than those values observed for protein enzymes.
Mutations within the nucleobase RNA sequences of ribozymes reduce the efficiency of cleavage. The most commonly found cleavage triplet is GUC. The effect on Kcat and Km differed depending on the mutation. Cleavage triplets with A as first or third nucleotide increased Km dramatically (less binding occurred), while there was little effect on the Kcat. Conversely, U had a large effect on Kcat, but has the same Km.
Therefore, different cleavage triplets have various catalytic efficiency (Kcat/Km).