Transcript Lecture 31

FCH 532 Lecture 31
Chapter 28: Nucleotide metabolism
Quiz on Mon (4/16): IMP synthesis-Purine synthesis
Quiz on Wed(4/18): Pyrimidine biosynthesis/regulation
Quiz on Friday(4/20): Ribonucleotide reductase
mechanism
Friday (4/20): extra credit seminar, Dr. Jimmy Hougland,
145 Baker, 3-4PM.
ACS exam has been moved to Monday (4/30)
Quiz on Final is scheduled for May 4, 12:45PM-2:45PM,
in 111 Marshall
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Proposed mechanism for rNDP reductase
Proposed reaction mechanism for ribonucleotide reductase
1.
Free radical
abstracts H from
C3’
2.
Acid-catalyzed
cleavage of the
C2’-OH bond
3.
Radical mediates
stabilizationof the
C2’ cation
(unshared
electron pair)
4.
Radical-cation
intermediate is
reduced by redoxactive sulhydryl
pairdeoxynucleotide
radical
5.
3’ radical
reabstracts the H
atom from the
protein to restore
the enzyme to the
radical state.
Catabolism of purines
•
•
•
All pathways lead to formation of uric acid.
Intermediates could be intercepted into salvage pathways.
1st reaction is the nucleotidase and second is catalyzed by purine nucleoside
phosphorylase (PNP)
•
Ribose-1-phosphate is isomerized by phosphoribomutase to ribose-5-phosphate
(precursor to PRPP).
Purine nucleoside + Pi
•
Purine base + ribose-1-P
Purine
Adenosine and deoxyadenosine are
notnucleoside
degraded by PNP but are deaminated by
phosphorylase
adenosine deaminase (ADA) and AMP deaminase in mammals
Figure 28-23 Major pathways of purine catabolism in
animals.
ADA
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Genetic defects in ADA kill
lymphocytes and result in
severe combined
immunodeficiencey disese
(SCID).
No ADA results in high levels of
dATP that inhibit ribonucleotide
reductase-no other dNTPs
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Figure 28-24a
Structure and mechanism of
adenosine deaminase. (a) A ribbon diagram of murine
adenosine deaminase in complex with its transition
state analog HDPR.
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Figure 28-24b
(b) The proposed
catalytic mechanism of adenosine deaminase.
1.
Zn2+ polarized H2O molecule
nucleophilically attacks C6 of the
adenosine. His is general base catalyst,
Glu is general acid, and Asp orients
water.
2.
Results in tetrahedral intermediate which
decomposes by elimination of ammonia.
3.
Product is inosine in enol form (assumes
dominant keto form upon release from
enzyme).
Purine nucleotide cycle
•
•
•
Deamination of AMP to IMP combined with synthesis of AMP
from IMP results in deaminating Asp to yield fumarate.
Important role in skeletal muscle-increased activity requires
increased activity in the citric acid cycle.
Muscle replenishes citric acid cycle intermediates through the
purine nucleotide cycle.
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Figure 28-25 The purine nucleotide cycle.
Xanthine oxidase
•
•
•
•
•
•
Xanthine oxidse (XO) converts hypoxanthine to xanthine, and xanthine to
uric acid.
In mammals, found in the liver and small intestine mucosa
XO is a homodimer with FAD, two [2Fe-2S] clusters and a molybdopterin
complex (Mo-pt) that cycles between Mol (VI) and Mol (IV) oxidation
states.
Final electron acceptor is O2 which is converted to H2O2
XO is cleaved into 3 segments. The uncleaved enzyme is known as
xanthine dehydrogenase (uses NAD+ as an electron acceptor where XO
does not).
XO hydroxylates hypoxanthine at its C2 position and xanthine at the C8
positon to produce uric acid in the enol form.
Figure 28-26a
X-Ray structure of xanthine
oxidase from cow’s milk in complex with salicylic acid.
N-terminal
domain is
cyan
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Central
domain is
gold
C-terminal
domain is
lavender
Mechanism for XO
1.
Reaction initiated by attack of enzyme nucleophile on the C8
position of xanthine.
2.
The C8-H atom is eliminated as a hydride ion that combines
with Mo (VI) complex, reducing it to Mo (IV).
3.
Water displaces the enzyme nucleophile producing uric acid.
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Figure 28-27 Mechanism of xanthine oxidase.
Figure 28-23 Major pathways of purine catabolism in
animals.
ADA
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Genetic defects in ADA kill
lymphocytes and result in
severe combined
immunodeficiencey disese
(SCID).
No ADA results in high levels of
dATP that inhibit ribonucleotide
reductase-no other dNTPs
Purine degredation in other animals
Primates, birds, reptiles,
insects-final degradation
product id uric acid which
is excreted in urine.
Goal is the conservation
of water.
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Figure 28-29 The Gout, a cartoon by James Gilroy
(1799).
Gout is a disease characterized by elevated levels of uric
acid in body fluids. Caused by deposition of nearly
insoluble crystals of sodium urate or uric acid.
Clinical disorders of purine metabolism
Excessive accumulation of uric acid: Gout
The three defects shown each result in elevated de novo purine biosynthesis
Common treatment for gout: allopurinol
Allopurinol is an analogue of hypoxanthine that strongly inhibits
xanthine oxidase. Xanthine and hypoxanthine, which are soluble, are
accumulated and excreted.
Catabolism of pyrimidines
•
•
•
Animal cells degrade pyrimidines to their component bases.
Happen through dephosphorylation, deamination, and
glycosidic bond cleavage.
Uracil and thymine broken down by reduction (vs. oxidation
in purine catabolism).
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Biosynthesis of of
NAD and NADP+
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Produced from vitamin
precursors Nicotinate
and Nicotinamide and
from quinolinate, a Trp
degradation product
Biosynthesis
of FMN and
FAD from
riboflavin
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FAD is synthesized from
riboflavin in a tworeaction pathway.
Flavokinase
phosphorylates the 5’OH
group to give FMN
FAD pyrophosphorylase
catalyzes the next step
(coupling of FMN to
ADP).
Page 1101
Biosynthesis of CoA
from pantothenate