biochem lecture 11 pt1 Flashcards
(176 cards)
1
Q
what are the two biosynthesis pathways for nucleotides
A
de novo synthesis, salvage pathway
2
Q
what are we focusing on
A
biosynthesis of the nucleotides, building blocks for DNA/RNA (not actual DNA synthesis)
3
Q
what is de novo synthesis
A
compounds synthesized from scratch/minimal components
4
Q
what are building blocks for de novo synthesis of nucleotides
A
metabolic precursors; parts of AAs, ribose-5-phosphate (byproduct of pentose phosphate pathway),
5
Q
what is ribose-5-phosphate important for
A
providing the structure that will become either the deoxyribose or ribose sugar in DNA and RNA
6
Q
what are salvage pathways
A
synthesis of nucleotides from scavenged components
7
Q
what are scavenged components
A
recycled free bases, nucleosides (present from breakdown of nucleic acids)
8
Q
what are 2 types of nitrogen containing bases
A
purines and pyrimidines
9
Q
describe purines structure
A
bicyclic
10
Q
describe pyrimidine structure
A
single ring
11
Q
what are the purines
A
adenine, guanine
12
Q
what are the pyrimidines
A
uracil, thymine, cytosine
13
Q
for purine de novo biosynthesis, what are the sources of parts of purine bicyclic ring structure
A
aspartic acid, formate, glycine, amine N of glutamine, Co2
14
Q
what is important precursor for purine de novo synthesis
A
5-phosphoribosyl 1-pyrophosphate (PRPP)
15
Q
what does de-novo synthesis of purine begin and end with
A
begins with PRPP, ends w/ inosinate (IMP) formation
16
Q
what is another important intermediate
A
inosinate (IMP) or inosinate monophosphate
17
Q
why is IMP formation important
A
from IMP synthesis, we have a bifurcation
18
Q
what is the purine bicyclic ring structure built off of in de novo synthesis
A
PRPP; serves as precursor for sugar
19
Q
what is IMP an important common intermediate for
A
synthesis of both AMP and GMP
20
Q
what do we have in each of these split, bifurcated pathways
A
an E source (GTP or ATP), and an amino group source (NH3)
21
Q
what do we have as the E source for AMP synthesis
A
GTP
22
Q
what do we have as the amino group for AMP synthesis
A
aspartate
23
Q
describe what’s in AMP synthesis
A
GTP as E source, aspartate provides amino group that’s part of adenylate structure for AMP
24
Q
what is E source for GMP synthesis
A
ATP
25
what is amino group source for GMP synthesis
glutamine
26
describe what's in GMP synthesis
ATP as an E source, glutamine as an amino source
27
what are both AMP and GMP
parent compounds
28
what happens to AMP and GMP
go through a set of phosphorylated rxns that will result in triphosphorylated forms
29
describe regulation of adenine and guanine nucleotide synthesis in bacteria
3 major feedback inhibition mechanisms/points of control
30
what are the 3 points of control in adenine/guanine nucleotide biosynthesis
PRPP syntethase, glutamine-PRPP amidotransferase, and adenylosuccinate synthesis
31
what do the 3 points of control represent
the 3 major feedback inhibition points
32
how do we balance the pool of different nucleotides in terms of ensuring you have appropriate levels of all 4/5 diff nucleotides
coordinated feedback inhibition
33
what does pyrimidine de novo synthesis involve
aspartate, PRPP, carbamoyl phosphate
34
what is the first enzyme in pyrimidine de novo synthesis pathway
aspartate transcarbamoylase or ATCase
35
how does purine pathway work
start off w/ PRPP, construct nitrogen containing base off the ribose moiety of that structure
36
how does pyrimidine pathway work
construct monocyclic ring first, middle of the pathway we attach PRPP or what eventually becomes ribose sugar
37
is there a difference between the purine and pyrimidine biosynthesis pathways
yup
38
what are the basic precursors for pyrimidine biosynthesis pathway
aspartate, PRPP, carbamoyl phosphate
39
basically how does pyrimidine biosynthesis work
we have the 6 member pyrimidine ring made first, that's gonna be attached to ribose-5-phosphate
40
what carbamoyl phosphate
intermediate used in first step of pyrimidine de novo pathway
41
what is important for synthesis of carbamoyl phosphate
bacterial carbamoyl phosphate synthetase
42
what does bacterial carbamoyl phosphate synthetase use in synthesis of carbamoyl phosphate
glutamine and ADP
43
describe bacterial carbamoyl phosphate synthetase
multi-subunit enzyme complex
44
what does bacterial carbamoyl phosphate synthetase use
substrate channeling
45
what is substrate channeling
helps retain/channel/keep intermediates that are part of synthesis step close to the enzyme, so that we don't lose those intermediates to diffusion
46
what happens if we lose intermediates to diffusion
can lessen efficiency of enzyme
47
when is substrate channeling particularly important
if intermediates are short-lived; if they decay or are unstable, we can lose those intermediates before they're utilized in this enzyme
48
how many active sites in bacterial carbamoyl phosphate synthetase
3 separate active sites
49
is ATCase same as the carbamoyl phosphate synthetase
no; that makes carbamoyl phosphate, so it's made by the time we get to first step in pyrimidine pathway
50
what is the first enzyme in the pyrimidine de novo synthesis pathway
ATCase (aspartate transcarbamoylase)
51
what kind of enzyme is ATCase
allosterically regulated enzyme
52
how do we know ATCase is allosterically regulated
because of the sigmoidal shaped curve
53
what is an important regulator of ATCase
CTP (cytosine triphosphate)
54
what does ATP do
reverses effects of CTP; serves as an antagonist, restores the same level of activity
55
what is a rightward shift indicative of
something inhibiting the enzyme (takes more of substrate to reach the same V)
56
what is CTP
feedback inhibitor of ATCase
57
what else is CTP
end product of one of the pyrimidines that are synthesized, so it serves as a feedback inhibitor
58
describe feedback inhibitory mechanisms in synthesis pathways
with synthesis pathways we're utilizing energy that's precious to the cell. when we have enough of end product, there are feedback inhibitory mechanisms that dampen/slow down/inhibit that process
59
what does ATP do
antagonistic effect on CTP; restores normal activity of ATCase
60
what happens if there's an imbalance of pyrimidine vs. purine pool and there's more ATP
ATP is purine, pyrimidines need to be balanced w/ the purine concentration in the cell, so more pyrimidines
61
what does the first enzyme in the pathway of pyrimidine de novo synthesis do
serves as point of regulation
62
what does CTP at the bottom do
serves as allosteric inhibitor (negative allosteric regulation of aspartate transcarbamylase)
63
what are 2 general types of nucleotide biosynthesis
ribonucleotides (RNA precursors), deoxyribonucleotides (DNA precursors)
64
what do ribonucelotides serve as
precursors of deoxyribonucleotides
65
what is made first in the de novo pathway
ribonucleotides
66
what enzyme carries out the series of redox reactions to go from ribose to deoxyribose
ribonucleotide reductase complex
67
what is deoxyribose to ribose
reduced form of ribose sugar
68
what does ribonucleotide reductase act on
ribonucleotide diphosphates (diphosphorylated forms of ribonucleotides)
69
what is ribonucleotide diphosphate being converted to
deoxyribonucleotide diphosphate
70
what does this mechanism culminate in
synthesis of dNDP or deoxy form of nucleotide diphosphate
71
basically what are we goin gfrom
OH group at the C2 carbon to H at that carbon
72
what serves as source of electrons
NADPH
73
what two pathways are important
one for transfer of electrons to NDP substrate, but also a resetting of the ribonucleotide reductase
74
what does ribonucleotide reductase have
two sulfhydryl groups, which correspond to specific cysteine residues within active site of enzyme
75
what is needed for enzyme to function
needs to be reset into the reduced form
76
what happens in the last redox step
convert NDP to dNDP, the thiol/sulfhydryl (SH) groups get oxidized and form a disulfide bridge (S-S)
77
describe path electrons take
NADPH to FAD, FAD to thioredoxin, ultimately electrons get transferred to ribonucleotide reductase
78
how does all this get set into motion
we're re-reducing these sulfurs that are part of the system
79
how do we re-reduce the sulfurs
NADPH (serves as feeder source of electrons), sequential redox, GSH (glutathione), so we have glutathione reductase. from glutathione reductase electrons are transferred to another protein glutaredoxin. from glutaredoxin we transfer electrons to ribonucleotide reductase to regenerate the reduce form of reductase (SH groups instead of S-S bridge)
80
what is this ribonucleotide reductase complex similar to
pyruvate dehydrogenase complex (idea of having to reset the system thru redox)
81
what do we need to do in order to allow for multiple rounds of reduction of NDP to dNDP
we have to regenerate these reduced thiol (SH) groups for each cycle
82
what do we end up doing to the sugar
replace 2' OH group w/ 2' H
83
do we deplete all of the ribose (turn it all into deoxyribose?0
no, we use some of that to make building blocks for RNA. but others will be used for generating deoxyribose form of sugar (DNA synthesis)
84
what kind of regulation does ribonucleotide reductase have
a complex kind of allosteric regulation
85
how many levels of control in ribonucleotide reductase
2 levels of control
86
what are the two levels of control in ribonucleotide reductase
primary regulation site, substrate specificity site
87
what does primary regulation site serve as
basic on-off switch for enzyme
88
how many regulatory subunits are there
2; R1 and R1
89
how many catalytic subunits are there
1; R2
90
what does active site have
thiols/cysteines with SH groups, where substrates bind
91
what is the point of having a combined primary regulatory site (on-off switch) and substrate specificity site
to provide balanced concentration of all the diff types of nucleotides or deoxynucleotides
92
what do we want in terms of the diff nucleotides
don't want to be able to carry out the reduction of just one type of ribonucleotide diphosphate; we want all the diff ones (A, T, Cs, Gs) to be reduced so that we have a balanced pool of those diff building blocks
93
what happens when we have an abundance of one type of nucleotide
effectively stimulates production of other types of nucleotides
94
what is the general idea behind this regulation
to have balanced pool of all the diff nucleotides
95
what is ribonucleotide reductase regulated by
dNTPs
96
why does dNTP serve as a feedback inhibitor of ribonucleotide reductase
this enzyme converts ribonucleotide diphosphates into deoxyribonucleotide diphosphates, when we have subsequent phosphorylation of those di-phosphorylated forms to generate triphosphorylated forms, when there's enough of the deoxy form of ATP, it will serve as a feedback inhibitor of enzyme
97
what serves as an activator for ribonucleotide reductase
having more of the ribonucleotide (ribo ATP form)
98
what effects do ATP vs dATP have on enzyme activity
opposing
99
what is the rule regarding substrate specificity
the nucleotide that serves as an allosteric regulator of substrate specificity is gonna favor reduction of other or alternate types of nucleotide diphosphates
100
what do dATP or ATP favor
reduction of UDP and CDP
101
what do dTTP or dGTP favor
reduction of GDP and ADP
102
what happens after we convert the ribo NDP to deoxy ribo NDP form by reductase
we need to go through additional phosphorylation
103
what generates the tri-phosphorylated forms
a series of kinases
104
what converts dCDP into dCTP
nucleoside diphosphate kinase
105
what happens to dUDP
converted to dUTP by the same enzyme
106
describe CTP and UTP
same thing, except CTP has an amino group constituent off the ring structure
107
what has to happen to go from dCTP to dUTP
deamination reaction, by enzyme deaminase
108
what does deamination reaction do
converts cytosine base into uracil base
109
what does dUTPase do
converts dUTP into dUMP
110
what is dUMP
important precursor for eventual synthesis of dTMP
111
what are the triphosphorylated forms of these nucleotides
what is used as eventual precursors for DNA and RNA synthesis
112
what are major products generated in degradation of purines and pyrimidines
uric acid and urea
113
what is an end product for degradation of GMP and AMP
uric acid
114
what do primates/mammals excrete
excess nitrogen
115
is nitrogen useful from an energetic standpoint
not really
116
what do primates generate from degradation of purines and pyrimidines
uric acid
117
but what do primates excrete the bulk of their nitrogen as
urea in urea cycle (as opposed to uric acid in purine degradation)
118
what are the two sources of excreted nitrogen
one from purines and pyrimidines, other from proteins and AAs that are degraded & used that pass thru urea cycle
119
do we excrete urea or uric acid
some uric acid, but majority of excreted nitrogen is in form of urea
120
who excretes uric acid
primates, birds, reptiles, insects
121
who excretes allantoin
most mammals
122
who excretes allantoate
bony fishes
123
who excretes urea
amphibians, cartilaginous fishes
124
who excretes ammonia
marine invertebrates
125
what is most abundant form of excreted nitrogen in primates, and where does that come from
urea; comes from degradation of proteins and AAs
126
important intermediate in catabolism of pyrimidines
methylmalonyl semialdehyde
127
what is methylmalonyl semi aldehyde degraded to
succinyl CoA (TCA cycle intermediate)
128
what is succinyl CoA
TCA cycle intermediate
129
what happens to carbon skeleton derived from catabolism of pyrimidines
can be shunted into TCA cycle
130
what are salvage pathways
pathways that scavenge or utilize free purine pyrimidine bases
131
how are these free bases released
thru degradation of nucleotides
132
do all nucleotides go through degradative pathways
not all, some can be salvaged and reused
133
how are free purine and pyrimidine bases released
via metabolic degradation of NTs
134
what are free purines used
salvaged and reused to make NTs
135
what is a major pathway for purines
adenine + PRPP --> generates AMP and PPi (pyrophosphate)
136
where else are there similar pathways
for pyrimidines in bacteria and mammals
137
what is a genetic disorder associated w/ defects in the salvage pathway enzyme
Lesch-Nyhan syndrome
138
Lesch-Nyhan syndrome is a result in defect in what enzyme
hypoxanthine-guanine phosphoribosyltransferase (HGPRT)
139
what enzyme is HGPRT
salvage patwhay enzyme
140
what is Lesch-Nyhan syndrome
almost exclusive to male children; profound mental retardation, self-mutilating behavior
141
what is seen in Lesch-Nyhan synddrom
elevated levels of de novo purine synthesis, so increase in uric acid
142
what other conditions are there
gout
143
how does gout arise
excess production of uric acid
144
what is gout
painful disease, affects joints and other tissues
145
why is gout created
b/c there are certain competitive inhibitors
146
what are competitive inhibitors
compounds/analogs, structurally similar to naturally occurring precursor/intermediate/end product
147
what are the competitive inhibitors in gout
enzyme xanthine oxidase, oxypurinol is its competitive inhibitor
148
what is allopurinol
inhibitor of one of the enzymes that leads to production of excess uric acid
149
what is allopurinol an inhibitor of
xanthine oxidase
150
what is oxypurinol
byproduct of action of xanthine oxidase on allopurinol drug; serves as competitive inhibitor of xanthine oxidase
151
what does oxypurinol do
inhibits XO
152
where else do we see structural analogs
in drugs, chemo
153
what is a common target of these chemotherapeutics
to target enzymes part of nucleotide biosynthesis pathway
154
why does it make sense that chemo targets these enzymes
tumor cells are v active, high rates of replication. one way to slow rate of proliferation is to block their ability to synthesize nucleotides, DNA and RNA, and precursors
155
what are some drugs designed for
to target diff points of nucleotide metabolism
155
what is one way to slow replication of tumor cells
block their ability to synthesize nucleotides, synthesize DNA/RNA, synthesis of precursors
156
what are two target points for nucleotide metabolism
enzyme thymidylate synthase, other enzyme is dihydrofolate reductase (DHFR)
157
what is one drug that targets these points
FdUMP
158
what is FdUMP
fluorine analog of the normal substrate for thymidylate synthase (dUMP)
159
what does thymidylate synthase do
converts dUMP to dTMP
160
what can happen to dTMP
become phosphorylated to generate dTTP, which is then used in DNA synthesis
161
what is another enzyme that is targeted by drugs
dihydrofolate reductase
162
what is dihydrofolate reductase
reductase; uses NADPH as an electron source, reduces folic acid
163
what is folic acid
an important precursor for certain nucleotides
164
what drugs target dihydrofolate reductase
methotrexate, aminopterin, trimethoprim
165
what are these drugs used for
to treat various types of cancers
166
what is FdUMP used for
to treat diff cancers
167
what does FdUMP serve as
suicide inhibitors
168
what are regular inhibitors
interact w/ enzyme via non-covalent interaction, binds and inhibits it. reversibly binding inhibitors; binding is reversible and can dissociate again
169
what are suicide inhibitors
utilized in the catalytic activity of the enzyme. rather than inhibitor being converted into product, it becomes covalently attached to the enzyme within its active site, forms this dead-end complex.
170
why is this dead-end covalent complex achieved
because there is a chemically reactive fluorine
171
what happens in normal conversion of dUMP to dTMP
normal 3 step process, produces dTMP at the end
172
what is FdUMP similar to
dUMP natural substrate for thymidylate synthase
173
what does FDUMP go thru
regular steps of catalysis, but reactive fluorine results in formation of this covalent dead-end complex
174
what happens after covalent dead-end complex
enzyme can no longer catalyze any reactions, because drug is covalently attached to the active site of the enzyme, so the enzyme is basically dead
175
why are they called suicide inhibitors
get used in the biochemical rxn catalyzed by the enzyme, that effectively results in an inactivated enzyme
