Rafferty Flashcards
what ions are used in oxidation and reduction
hydride ions = H- or H+ + 2e-
what is the structure of NAD
Nicotinamide adenine dinucleotide (phosphate) NAD(P)
dinucleotide 2 ribose rings connected by 2 phosphates
1st ribose is connected to a nicotinamide ring, where the chemistry occurs
2n ribose is connected to an adenine ring
where does hydride transfer occur in NADH
C4 of nicotinamide ring (one opposite to bond with ribose)
what is the difference between NAD+ and NADH
NAD+ is the oxidised form it has 1H on the C4 of the nicotinamide ring - the +ve charge is distributed over the ring
NADH is the reduced form it has 2H on the C4 of the nicotinamide ring - it has lost the charge and the plane is disrupted, N has a lone pair
what is the mechanism of hydride transfer of NADH
the lone pair from the N moves around the ring to the bond to the 2nd H on the C4 of the nicotinamide ring which leaves a hydride ion, attacking a C breaking a carbonyl (C=O)
what is the difference between NADPH and NADP+
an extra P on the 2’ of ribose of adenine
eg. indicates biosynthesis
what is critical to the hydride transfer in NADH
the angle of approach due to stereospecificity
distance ~ 3-3.3A
what is acp
acyl carrier protein
attached via phosphopantetheine arm
covalently linked to Ser residue on ACP
beta-ketoacyl reductase BKR
1st reductive step
Rossman fold
NADPH dependant
tetrameric
what is Rossman fold important for
substrate binding
active site of BKR
conserved lysine (stabilised -ve O on enolate anion) and tyrosine (4 residues apart) near NAD nic ring
active site of ENR and difference between BKR (and why)
conserved lysine and tyrosine are 8 residues apart - the substrate is different
ENR enoyl reductase
final reductive step
NADH dependant
also tetrameric
why is ENR a good drug target
it is in bac. but differnet in euk. so it is a possible anti-biotic
how to draw ENR mechanism
arrow starts from bond between NADH and H (hydride ion)
what is tricolsan
substrate intermediate mimic for ENR (Enolate anion)
tautomers
e- swap between c=c-o and c-c=o
constantly swap
balance changes depending on condition
what happens when ENR is inhibited
fatty acid synthesis is not completed
membrane disrupted
cell lysed
ENR inhibitors how do they work
diazaborine - competitive inhibitor, forms covalent bond for nic ring, resistance developed quickly (single base change needed), boron toxic
tricolsan - competitive inhibitor, substrate intermediate mimic (enolate anion)
beta hydroxyl dehydratase BHD
removes H2O forms double bond
how does fatty acid chain elongation occur
via successive addition of 2C unites the S-acyl primed acyl carrier protein
what is the difference between tautomers and resonance structure
tautomers are 2 distinct structures which are in an equilibrium
they convert rapidly between each other (tautomerisation) often involved the movement of a H+ and its accompanying electrons
resonance is 2 representations of the configuration of e- in one structure
the 2 structures do not exist individually
what kind of reaction is the breakage of the phosphodiester bond (2 answers)
hydrolysis - breaking with water
SN2 - substitution, nucleophile, bimolecular
how does the OH- (derived from a water molecule) nucleophile attack the phosphodiester bond
attacks the phosphorus of the phosphate ion
attacks from the bottom with the leaving group at the top because it is going to invert the configuration around the phosphorus (means careful arrangement of substrate is needed)
how can the RNA phosphodiester bond be self cleaved
what does this result in
in alkaline conditions - a base can abstract the proton from the 2’ -OH making a O- nucleophile which can attack the phosphorus
results in a breakage of the 5’ phosphodiester bond using a 3’-2’ cyclic intermediate in which one bond is broken to give a 3’ or 2’ phosphodiester bond (random)
how does a restriction endonuclease couple to DNA
non-specific binding to stand
linear diffusion - moves along strand like train tracks
specifically binds
couples (bends DNA) –> catalysis
structure of EcoRV and EcoR1
cup shape for substrate
proline, Asp, Asp/Glu, Lys - split into 2 motifs PD and D/ExK
how do RNases utilise metal ions to enable the catalytic process
they stabilise the negative charges on the pentavalent intermediate
what metal ions are used in RNases
Mg2+ and Mn2+
MN more effective but low abundance
Ca2+ seems to have an inhibitory effect
