BIOINORGANIC CHEMISTRY A Short Course Second Edition

280 GROUP I AND II METALS IN BIOLOGICAL SYSTEMS: GROUP II

Researchers in the fi eld continued to study the hammerhead ribozyme –
substrate complex by biochemical and spectroscopic means. Three papers of
the many published between 1999 and 2001 will be used here to illustrate the
types of experimental results and to defi ne conclusions based on these. In a
Biochemistry paper published in 1999, Hershlag and co - workers continued
their biochemical study of the hammerhead system and identifi ed a hammer-
head ribozyme metal ion binding site that appeared to interact with the ham-
merhead substrate ’ s cleavage site even though these sites are ∼ 20 Å apart in
the X - ray crystallographic ground state structures.^45 The research studied two
important hammerhead sites by replacing specifi c phosphate oxygens with
sulfur, determining the effect on catalytic cleavage rate for the replacements
and then determining if the thiophilic metal ion Cd 2+ could rescue catalysis.
Substitution of specifi c phosphodiester oxygens by sulfur dramatically lowers
Mg2+ affi nity for the site resulting in decreased catalysis. Addition of more
thiophilic metals such as Cd 2+ or Mn 2+ is known to “ rescue ” catalysis.
The two proposed metal binding sites were (1) the ribozyme P9/G10.1 site,
located at the junction between stem (helix) II and the conserved catalytic
core, and (2) the substrate P1.1, scissile phosphate, site (see Figure 6.18 ).
For the HH16 hammerhead construct used in this research the specifi c resi-
dues at the fi rst site would be A 9 /G 10.1 , and those at the second, cleavage, site
would be G 1.1 /C 17. The specifi c phosphate oxygen atoms binding the metal at
both sites would be the nonbridging prochiral pro - R p phosphate oxygens, the
same sites that were occupied by metal ions in the PDB: 301D X - ray crystal-
lographic structure. For instance, looking at Table 6.4 for PDB: 301D, one fi nds
that the bonding distance from Mg 2+ at site 6 to the pro - R p phosphate O of
A1.1 is equal to 2.43 Å. It had been shown previously and confi rmed in this
study that replacement of the pro - R p phosphate oxygen with sulfur at either

Figure 6.18 “ Rescue ” sites in the HH16 hammerhead ribozyme construct.

N A^9

N

HN

N

NH 2

HO O

O

O

O

(S)O PO

HN OH O

N

HN

N

O

H 2 N

2' 3'

5'

G10.1

Me2+

3'

Rp

5'

C 17

N

HN

O NH 2

HO O

O

O

O

(S)O PO

HN OH O

N

HN

N

O

H 2 N

2' 3'

5'

G1.1

Me2+

3'

Rp

5'

B

P1.1 site

A

P9/G10.1 site

7