Ribozymes
In vivo self-splicing introns are not catalyst, but true catalyst can be constructed from them,
which function in vitro.
Ribozymes are much like proteins, and they have a 3-D structure that is required for their
function.
People who study ribozymes measure many of the same properties as people who study enzymes.
(kcat, KM, and Vmax).
For ribozymes, substrate binding and/or product release tends to be the slow step, while for
enzymes the chemistry is the slow step.
An enzyme that catalyzes a similar reaction to a ribozyme is almost always a better catalysts
(kcat/KM). Proteins have more functional groups.
Ribozymes are slower at catalyzing the chemistry and have more problems with substrate/product
binding/release.
People have “selected” ribozymes in vitro that catalyze a number of reactions.
People have also “selected” DNA catalysts in vitro. They tend to be poorer catalysts than
ribozymes (2’ OH’s play an important role in the folding and catalytic properties of ribozymes).
The Chicken and the Egg of Evolution
Which came first proteins and DNA?
Without DNA there is no way to direct protein synthesis, but DNA can not synthesize itself.
In the first living organism it is believed that RNA (or even a precursor of RNA) was both
the genetic information and the catalysts (a self-replicating RNA).
How many reactions did these RNA organisms catalyze?
It is believed that they had a diverse biochemistry.
It is believed they used all of the base containing cofactors and molecules:
NAD, NADP, CoA, and etc.
The base was a “handle” for these ribozymes.
If the organisms were that complex, they almost certainly had a membrane. It makes no sense
to produce NADH or NAD if it is just going to float way.
Conformation Change and the Hammerhead Ribozyme
In the crystal structure of the ribozyme, the bond that is cleaved is not in the proper geometry for
in-line attack.
However, the G-8 2’ OH is oriented to undergo the transesterfication reaction with in-line attack.
W. G. Scott
JMB, 2001, 311
pg. 989-999
However, this happens extremely rarely. Why? What has to happen for a reaction to occur?
Orbital Steering and the Hammerhead Ribozyme
H-bonds prevent the lone pairs from interacting with the unoccupied d orbitals of the P. The H
bonding network is constructed so they are pointed in opposite directions of the d orbitals. This
prevents the reaction from occurring
Calculations suggest that the orbital steering decreases the rate of reaction by at least 1,000 fold.
This suggests that orbital steering could increase the rate of reaction over 1,000 fold (i.e. if H
bonds were set-up to force interactions between the d orbitals and a lone pair).
It is believed the enzymes also use orbital steering to catalyze reactions.
From:
Stepan Sklenak, Lishan Yao,
Robert I. Cukier, and Honggao Yan
J. Am. Chem. Soc.; 2004;
126(45) pp 14879 - 14889
More Base Modifications
Thiolation of bases is a common type of modification.
S O NH2 NH2
N
NH N N
NH
S N N N
O N S N SH
R R
R R
In E. coli, all sulfur containing modifications are dependent upon iscS.
iscS knock outs are also deficient in some Fe-S cluster containing proteins. iscS is also involved
in Fe-S cluster biosynthesis (as well as some other S containing molecules).
IscS is a cysteine desulfurylase. In E. coli, there are three IscS like proteins; the role of the other
two is not clear.
IscS catalyzes the following reaction:
IscS + cysteine IscS•S + alanine
Where the sulfur from the cysteine is attached to a cysteine in IscS forming a persulfide.
IscS S
S-
IscS and PLP
IscS is a PLP dependent enzyme.
PLP is a common cofactor and is used as an “electron sink”.
Fig. 26-1
PLP is covalently attached to Lys of enzymes using a Schiff base.
The Proposed Mechanism of PLP Dependent Cysteine Desulfurylases
B: BH BH
H COO2-
-
HS COO2-
COO2 HS
HS NH+
IscS (Cys)-SH NH+
NH+ IscS (Cys)-S-
R R’ R R’
R R’ ..
N R’’
H N+ R’’
N+ R’’
H
H
BH H
B: COO2-
COO2- COO2-
:NH NH+ NH+
IscS (Cys)-S SH IscS (Cys)-S SH
R R R R’
R’ R’
N+ R’’ N+ R’’ N+ R’’
H H H
Biochemistry; V 33; No 15; 1994; pg 4714-4720
PLP and Transaminations (Part 1)
note all of the steps are now shown here, but we will talk about them all, and you will have to be able to draw them all.
Fig 26-2
PLP and Transaminations (Part 2)
the reverse of the other slide
Can you draw it?
PLP and Decarboxylations
Arginine decarboxylase
+ Enz-PLP
-O O O- BH H H + Enz-PLP
C + CO2
O C C R HC R
HC R CH R N NH2
NH2 +HN
O-
2-O PO
kcat = 1375 s-1
3
O-
2-O PO
3
N
H
N
H+
Enzyme Families And Signature Sequences
Class I aminoacyl-tRNA synthetases could be called an “enzyme family”.
Sometimes enzymes in the same family catalyze different reactions, but they almost always
share some chemistry.
Enzymes in a family almost always share a signature motif. All Class I aminoacyl tRNA
synthetases contain a HIGH motif.
These motifs are almost always important for the function of the enzyme.
Sometimes things get a little confusing:
There is a family of enzymes that catalyze different reactions, but all contain an SGGXDS motif.
They all catalyze adenylations (of different substrates). They are called the PPi synthetase family.
Aminoacyl-tRNA synthetases do not contain this motif and are not a member of the family even
though they catalyze adenylations.
There are also structural families. These are proteins that share the same 3-D structure. The same
structure can be obtained by proteins that have very little sequence similarity (i.e. the aldolases).
Thiolated Base
S O NH2 NH2
N
NH N N
NH
S N N N
O N S N SH
R R
R R
SAM and Radical Formation
S-adenosylmethionine radical enzymes.
Bioorg Chem. 2004 Oct;32(5):326-40.
SAM and Radical Abstraction
Can the same chemistry be used to make 2-thioadenosine?
S-adenosylmethionine radical enzymes.
Bioorg Chem. 2004 Oct;32(5):326-40.