Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

256 Chapter 12
self-splicing reaction requires no protein-based molecules, but requires two
cofactors: a divalent metal ion such as Mg 2þ ; and a guanosine monomer,
which can either be phosphorylated or bear a free 5 0 -hydroxyl group.
The mechanism of this self-splicing reaction has been elucidated, and is
shown in Figure 12.1. The RNA precursor is able to bind the guanosine cofactor
in the presence of Mg 2þ ions. The 3 0 -hydroxyl group of the guanosine cofactor
then attacks the phosphodiester linkage at the 5 0 cleavage site, displacing
a free 3 0 -OH and becoming covalently attached to the RNA molecule. It is
thought that the Mg 2þ acts as a Lewis acid to assist the leaving group properties
of the departing 3 0 -OH. The cleaved RNA strand remains non-covalently bound
to the ribozyme through a six-base complementary ‘guide sequence’, whilst a
conformational change brings G 414 into the guanosine binding site. The free
uridine 3 0 -OH then attacks the G 414 –U phosphodiester bond, forming the new
phosphodiester bond of the shortened RNA, and releasing the ribozyme which
terminates in G 414 .
How eVective is this ‘ribozyme’ as a catalyst The site of cleavage and
sequence selectivity of the Tetrahymena ribozyme is very high, and the rate
constant calculated for the transesteriWcation step is an impressive 350 min 1 ,
some 10 11 -fold greater than the rate of the corresponding uncatalysed reaction.
HO
O
N
N
G 414 -U
O
NH
N NH 2
3'
strand
cleavage
G 414 -U
3'
HO OH
5'
O −
C-U-C-U-C-U O
P O
G-G-G-A-G-G-A O
5'
C-U-C-U-C-U-OH
G-G-G-A-G-G-A-G
conformational
change
O
O
N
N
O
NH
N NH 2
ligation
O
O
N
N
O
NH
N NH 2
new phosphodiester bond HO OH
5' C-U-C-U-C-U-U
G-G-G-A-G-G-A-G
3'
5'
O OH
O
O P
−
C-U-C-U-C-U
O
OH
G-G-G-A-G-G-A-G
U
3'
Figure 12.1 Mechanism of self-splicing reaction of Tetrahymena ribozyme.