Lecture 15 Antibiotics and RNA catalysts

Lecture 15 Antibiotics and RNA catalysts

11.10.15 - Lecture 15- Antibiotics and RNA catalysts
R
Penicillin antibiotics-inactivated by beta-lactamase's ability
to perform a fast deacylation of the
inhibitor-enzyme adduct
S
R
N
O
O
CO2
R
H2O
S
O
HN
O
CO2
Enz-Ser
OH
Enz-Ser
Inhibitors of beta-lactamases
R
Enz-Lys NH2
O
R
O
HN
O
R
SO2-
Enz-Ser
-
O
CO2
OH +
O
Enz-Ser
NH
O
O2S
O
H2N
CO2
-
Enz-Ser
Stable enzyme adduct
builds up
O O
S
HN
OH
R
O
Enz-Ser
R
Lys-Enz
NH
HN
O
CO2
Enz-Ser
CO2
Enz-Lys
O O
S
R
N
O
CO2
OH
Enz-Ser
O O
S
HN
OH
S
Deacylation
CO2
Using an alternative leaving group-
R
O
O
N
O
R
OH
CO2
CO2
OH
O
R
OH
+
HN
O
HN
O
Enz-Ser
O
O
CO2
Enz-Ser
In general,beta-lactam antibiotics
are not affected by drug pumps as their
target resides in the periplasmic space
Targeting D-Ala D-Ala ligase
O
ONH2
O
O
O
O P O P O
O- AMP
O+
-
O
O
O P O-
NH2
O
H2N
-
+
H2N
H2N
P
O
H2N
O
O-
O
-
O
-
O-
H
N
O
H2N
Design of an inhibitor of alanine ligase
-inhibition is dependent on presence of ATP
- off rate t1/2 ~17 days
- koffINH << koff PDT, 108-109 slower off rate
-example of slow binding/slow off rate inhibitor
OO P OO
O
O
H2N
CO2Enzyme performs slow
transphosphorylation in
presence of ATP
O
CO2-
P
O
O P OO-
Better TS analog
Previously discussed antibiotic mechanisms targeting D-Ala D-Ala
-transpeptidase inhibitor
-inhibitor of alanine racemase
-inhition of D-Ala ligase
Vancomycin-a natural product 'receptor' which specifically binds to D-Ala-D-Ala
via a network of H-bonding and blocks transpeptidase
Vancomycin Resistance
-bacteria use lactic acid instead of D-Ala
-ester linkage is much less favorable, repulsive
interactions between oxygen lone pairs, loss of H-bond
O
D-Ala
O
O
O
O-
NH2
O-
OOH
Lactic acid
Loss of H-bond and
introduction of unfavorable
interaction
RNA catalysts
-RNA can carry out catalysis
-most often dependent on presence of
Magnesium
Self splicing RNA in tetrahymena
General reaction3'
YYUpN + G-OH3'
G-OH
YYU-OH3' + GpN
(Y = pyrimidine base)
5'
Intron splicing
cucucu *
AGGGAGG
5'
U
U
U
G*uaaggu-OH 3'
cucucu-OH 3'
AGGGAGG-UUU*G-OH3'
5'
cucucu*uaagu-3'
G*uaaggu-OH 3'
(ligated exons)
+
3' HO-G
O-G
5' G*UUU-OH
5' G*UUU
AGGGAGG
AGGGAGG*
CIVS
Catalytic RNA
-in order to be catalytic, the catalyst molecule
must remain unchanged at the end of the reaction
3' HO-G
RNA which ligates to DNA substrateRNA ligaseACUCG-OH 3'
ACUCG-OH 3'
CCCTCCTpA3TA3TA
CCCUCCUpA3UA3UA
GGGAGG-5'
GGGAGG-5'
CCCCpC
GGGAGG-5'
C4
3' HO-CpG
CCCCC-OH
GGGAGG-5'
C6
ACUCGpA3TA3TA
ACUCGpA3UA3UA
S
GGGAGG-5'
primer sequence
enzyme can be amplified
selectively since only catalytic
RNAs are selected for
GGGAGG-5'
Amplification of starting RNA with RT rxn, PCR, and
in vitro transcption with T7 polymerase. Sequence diversity generated by error prone
PCR conditions or using an error
prone DNA polymerase
3' HO-G
RNAs with increased catalytic activities
GGGAGG-5'
Starting RNA
kcat - 2x10-4 min-1
Km ~7 M
After selection
kcat - 0.007 min-1
Km ~2 M
+ C4 + C6
(catalytic RNA unchanged)
(catalyzes disproportionation reaction)
RNA
RT
DNA
PCR
DNA
In vitro
transcription RNA
T7 Promoter
reverse
transcriptase
DNA polymerase
(error prone)
(amplification step)
T7 polymerase
(amplification step)
An RNA ligase selected from
randomized sequence
-randomize 220 bases
-library will be incomplete (4220 potential members possible); must assume many potential solutions
Using this strategyan RNA enzyme was discovered with
a t1/2 of 5 minutes vs. 33 years compared
to the uncatalyzed reaction
(107 rate acceleration)