Rafferty Flashcards
What is borohydride used for in chemistry?
- routine reductant and source of hydride ions
Why is there a problem with using borohydride in biological systems?
- boron can be harmful
- borohydride highly nonspecific
What are used as alts to borohydride in biology, and why are they better?
- cofactors, such as NAD(P)H
- much more gentle and precise
What part of NAD(P) is where the important chem happens?
- nicotinamide ring
What position does hydride transfer occur on in NAD(P)?
- C4 position on nicotinamide ring
What is the charge on NAD(P) and where is it located?
- +ve charge distributed over ring
- bit net overall charge is -ve, due to 2 phosphates
What diff sources can NAD(P) come from?
- nicotinic acid digested
- nicotinamide from breakdown of Trp
- nicotinamide mononucleotide –> ring system stuck onto ribose, in most systems take and use NAD pyrophosphorylase, which uses ATP to add on adenine-ribose-PO4- and add on 2 PO4- to make complete NAD molecule
What is the diff in shapes of NAD and NADH?
- NAD planar, and NADP not
How do NAD and NADH act as oxidoreductases?
- DIAG*
- hydride from NADH transferred to C of planar target molecule and one of bonds to O broken
- O tries to pull off proton from another functional group on surface of enzyme
- reaction also works in reverse using NAD
How is hydride ion stereospecific?
- enzyme differentiates between 2 hydrogens (pro-R or pro-S) on C where hydride transfer occurring
- transfers to 1 particular face on nicotinamide ring –> dep on whether sitting above or below substrate
How is distance of approach of hydride ion critical to its transfer?
- NAD(H) cofactor and molecule to which hydride transferred, or from which received typically at approx VDW contact distance (≈ 3-3.5Å)
How is angle of approach of hydride ion critical to its transfer?
- nucleophile attacks C=O at 107° angle, as orbitals lean away from node DIAG
- vertical is Burgi-Dunitz angle
- horizontal is Flippin-Lodge angle
What are FA synthetases (FAS) and what is their role?
- complex enzyme system
- carry out de novo biosynthesis
What are the 2 types of FAS and where are they found?
- type I have catalytic domains on 1 or 2 really big polypeptides –> in vertebrates, yeast and some bacteria
- type II have discrete polypeptides catalysing each enzymatic step –> in plants and many bacteria, inc E. coli and M. tuberculosis
What does chain elongation involve, and in what systems does it occur?
- successive addition of 2C units to S-acyl primed acyl carrier protein (ACP)
- occurs in type I and II systems
What occurs during the FA elongation cycle?
- DIAG*
- R group gets longer and longer
- C=C bond red by enoyl reductase and NAD(P) –> NAD(P)+
- release of FA products
- another molecule joined by β-ketoacyl synthetase, prod C=O
- ketone group on C3 red to hydroxyl by β-ketoacyl reductase and NAD(P)H –> NAD(P)+
- removal of water by β-hydroxyacyl deHydratase
What is the role of E. coli ACP, and how is the acyl group attached?
- transport protein that carries growing acyl chain
- attached via phosphopantetheine arm covalently linked to Ser32
What does the crystal structure of E. coli acylated ACP show?
- 4 helix bundle
- lipophilic cavity –> where put growing acyl chain, but can’t stay there all the time, as needs to do chem and undergo elongation
- ligand/acyl group exists in “bound”/buried and “unbound”/non-buried forms
How is the growing FA chain bound by ACP?
- binding pocket expands to accom growth
- binding stabilises ACP
- FA chain and phosphopantetheine arm adopt no. of diff binding modes
What is the role of beta keto reductase (BKR)
- catalyses 1st reductive step of FA elongation cycle
What is BKR dep on?
- NADPH
What is the structure of BKR?
- tetrameric –> 4 active sited indep
What family is BKR a member of?
- short chain deHase-reductase (SDR) family of NAD/NADP dep oxidoreductases
What common critical feature do SDR family of enzymes have?
- Rossman fold and S…YxxxK motif