[11/22/09]

 Linda  Geng,  TA  

HUBIO  514:    BIOCHEM  REVIEW  for  “Nucleic  Acid  Metabolism”  

Let’s start with the basics…

• Metabolism:  chemical  reactions  inside  living  organisms  to  maintain  life.  Yes, it’s important!  

o Anabolic:  use  energy  to  make  nucleic  acids,  proteins,  etc.  

o Catabolic:  breaking  things  down  
• Nucleic  acids:    chains  of  nucleotides.  DNA  and  RNA.  
• Nucleotides:    nitrogenous  base  (A,  T,  C,  G,  U)  +  sugar  (ribose  or  deoxyribose)  +  phosphate  (1-­‐3)  
o Ex.)  ATP,  dATP,  ADP,  AMP,  dTTP,  dUMP,  etc.  
o RNA  uses  ATP,  GTP,  CTP,  UTP      vs.      DNA  uses  dATP,  dGTP,  dCTP,  dTTP  
• Nucleosides:    nitrogenous  base  +  sugar  

 
• Purines  =  A,  G     Pyridimidines  =  C,  U,  T  

Now for the pathways…

• How  nucleic  acid  metabolism  all  interrelates:    [make,  recycle,  degrade]    Purines  vs.  Pyrimidines  
PURINE  BIOSYNTHESIS  (anabolism):    making  of  purines  A  &  G
• What  are  purines  made  from?  3  amino  acids  &  a  folate  derivative
• How  are  they  made?
o PRPP  +  Glutamine  (Common  pathway)    IMP
o IMP  can  then  be  turned  into  AMP  &  GMP
• How/where  is  this  pathway  regulated?  Feedback  inhibition
 
 
 
 
 
• What  drug  targets  this  pathway?    6-­‐
mercaptopurine  
o Activated  by  HGPRT  to  form  6-­‐MP  nucleotide  and  acts  as  fake  purine  nucleotide  
o Results  in  feedback  inhibition  of  all  three  critical  steps  (1st  of  common  &  1st  of  both  branches)  
• Where  do  all  the  ingredients  of  purine  nucleotide  biosynthesis  come  from?    
o The  sugar  is  from    PRPP    ribose-­‐5-­‐phosphate    pentose  phosphate  pathway  
o Nitrogenous  base    glutamine    glutamate  
o Nitrogenous  base    N10-­‐formyl-­‐tetrahydrofolate    folic  acid  
o Nitrogenous  base    glycine    serine    glycolysis  
o Phosphates  are  put  on  by  specific  kinases    

PYRIMIDINE  BIOSYNTHESIS  (anabolism):    making  of  pyrimidines  C,  U,  T  

• What  are  pyrimidines  made  from?  carbamoyl  phosphate  &  aspartate  
• How  are  they  made?  
o Glutamine  +  stuff    carbamoyl  phosphate    orotate    OMP    UMP  
o UMP    UDP    UTP    
o UTP  can  be  turned  into  CTP  in  one  step  
o dTTP  is  a  little  more  complicated:    UDP    dUDP    dUTP    dUMP    dTMP    dTTP    (see  next  pg.)  
• What  step(s)  can  be  screwed  up  in  disease?  mutation  of  enzyme  for  orotateUMP  =  orotic  aciduria  
o Build  up  orotate  =>  excrete  tons  of  orotic  acid  in  urine  
o Bladder  stones,  mild  mental  retardation,  megaloblastic  anemia  
o Patients  need  to  eat  pyrimidines  ‘cause  they  can’t  make  ‘em!  
o Rx:    oral  uridine  
• How  is  this  pathway  regulated?    UTP  inhibits  first  3  steps.    Again,  feedback  inhibition.  

• Where  do  all  the  ingredients  of  pyrimidine  nucleotide  biosynthesis  come  from?    
o The  sugar  is  from    PRPP  
o Nitrogenous  base    carbamoyl  phosphate  +  aspartate  
o Phosphates  put  on  by  specific  kinases  
 
 
 
• Thymidine  synthesis  is  special:      
o Both  CDP  and  UDP  can  be  precursors  of  dUTP  
o dUMP  is  the  substrate  for  the  enzyme  thymidylate  synthetase  
 
 
 
 
 
 
 
 
 
 
 
 
 
• What  two  enzymes  must  work  together?  Thymidylate  
synthase  +  Dihydrofolate  reductase  (DHFR)  
• What  drugs  inhibit  the  synthesis  of  dTMP?              
o 5-­‐fluorouracil:    converted  to  fDUMP  and  used  as  
fake  substrate  that  inhibits  thymidylate  synthase  
o Methotrexate:  inhibits  DHFR  
 

• What  is  another  way  to  generate  dTMP?    Thymidine  kinase  

(TK)  is  an    enzyme  that  adds  a  phosphate  to  deoxythymidine  
to  make  dTMP  
o Deoxythymidine  is  from    degradation  of  DNA  from  
  food  &  other  dead  cells    [this  is  PYRIMIDINE  SALVAGE/recycling]  
 o The  dUMP  route  (above)  is  used  by  cells  normally  and  it’s  making  enough  dTMP  for  DNA  
repair/maintenance  
o But  when  cells  divide  and  DNA  replication  is  needed,  TK  is  expressed  to  increase  dTMP  supply  
o After  division,  return  to  normal  and  get  rid  of  TK  (known  as  a  “salvage  enzyme”)  
o We  have  our  own  TK,  but  so  do  viruses!    
o What  drugs  target  viral  TKs?      
 Acyclovir:  for  Herpes  (can  take  
systemically  b/c    specific  for  viral  TK)  
 IUdR  (iododeoxyuridine):    for  corneal  
Herpes  (put  topically  b/c    affects  
human  TK)  
 AZT  (zidovudine)  &  ddI  
(dideoxyinosine):    for  HIV  (can  be  used  
by  human  TK,  but  preferred  by  HIV  TK)  
 
 
 
 
 
 
 
DEOXYNUCLEOTIDES:  
ATP,  GTP,  UTP,  CTP  ribonucleotides  can  be  directly  used  for  RNA  synthesis,  but  need  to  remove  the  2’  OH  group  
on  sugar  to  generate  the  deoxyribonucleotides  needed  for  DNA  synthesis  (dATP,dGTP,  dTTP,  dCTP)  

 
• What  enzyme  works  on  all  four  ribonucleotides  to  pluck  off  the  –OH  group?  Ribonucleotide  reductase  
o CDP  turns  into  dCDP,  GDP  turns  into  dGTP,  ADP  turns  into  dADP  
o UDP  turns  into  dUDP,  which  then  goes    dTTP  (see  above  on  thymidylate  synthase)    
• Works  together  with  thioredoxin  reductase
• NADPH  is  what  provides  the  reducing  power
• Complex  network  of  cross-­‐regulation
• General  enzyme  activity  sites:
o Activated  by  ATP
o Inhibited  by  dATP  (*see  ADA-­‐SCID)
• Substrate  specificity  sites:
o Help  keep  balance  among  4  species
 
 
 
• What  disease  is  associated  with  the  dysregulation  of  this  enzyme?    ADA-­‐SCID  
 o Adenosine  DeAminase  enzyme  is  the  first  enzyme  in  the  break-­‐down  of  
adenosine  nucleotides  (*see  degradation  pathways  later)  
o Deficiency  of  ADA    more  dATP  block  ribonucleotide  reductase    
  no  DNA  synth    reduced  proliferation  of  immune  cells  (B  &  T  cells)  
o Build  up  of  nucleotide  metabolites  also  toxic  to  developing  immune  cells  
o Accounts  for  ~  25%  of  SCID  (severe  combined  immunodeficiency)  cases  

o Rx:    inject  ADA,  bone  marrow  transplant,  gene  therapy  /  stem  cells  
 
 
 
 
 

 
 
 
 
NUCLEOTIDE  CATABOLISM:    breakdown/degradation  of  nucleotides  
PURINE  DEGRADATION:  dietary  and  cellular  nucleic  acids  constantly  being  broken  down.  Many  of  the  resultant  purines  
are  catabolized  and  excreted  out  of  the  body  as  uric  acid.  

• What  are  the  starting  molecules  for  

purine  catabolism?  Adenosine  &  
guanosine  
• What  is  different  about  adenosine  &  
guanosine  breakdown?    
 o Adenosine  gets  deaminated  to  
inosine  
o Guanosine  gets  deribosylated  
to  its  free  base  form  guanine  
• What  is  the  intermediate  product  of  
purine  catabolism?    Xanthine  
• What  is  the  end  product  that  is  
excreted  out  of  the  body?    Uric  acid  
• What  disease  is  due  to  excess  uric  acid  
accumulating  in  the  body?    Gout  
• Why  is  the  buildup  of  uric  acid  a  problem  in  the  body  (why  does  it  cause  gout)?    it’s  insoluble,  so  forms  crystals  
in  joints  (especially  where  it’s  cooler  temperate,  like  extremities)  =  ouch!  pain  in  joints  of  fingers/toes    
o Hyperuricemia  =  increased  blood  levels  of  uric  acid  
o Either  due  to  (a)  more  purine  degradation  or  (b)  decreased  uric  acid  clearance  
o 80%  of  gout  cases  due  to  problems  in  renal  excretion  
o Rx:  Allopurinol  is  xanthine  oxidase  inhibitor  that  prevents  conversion  of  xanthine  to  uric  acid  

o Rx:  avoid  ingestion  of  items  rich  in  purines  (that  includes:      )  
• What  is  the  role  of  purine  catabolism  in  reperfusion  injury?    
o Ischemia  (lack  of  O2)    direct  cell  injury  or  indirect  injury  upon  reperfusion  (blood  returns)  
o Reperfusion  injury  seen  in  myocardial  infarctions,  strokes  ,  tissue  transplantations  
o During  ischemia,  ATP  is  not  regenerated  
o Xanthine  oxidase  enzyme  catalyzes  oxidation  of  hypoxanthine  
to  xanthine  and  then  also  catalyzes  oxidation  of  xanthine  to  
uric  acid  
o Ischemia  causes  the  cleavage  of  xanthine  oxidase  to  a  form  
that  produces  superoxide  free  radicals    oxidative  damage  
o Reperfusion  =  blood  supply  returns  after  period  of  ischemia  
o Reperfusion  injury  =  absence  of  O2  and  nutrients  from  blood  
creates  condition  in  which  restoration  of  circulation  results  in  inflammation  and  
oxidative  damage  through  induction  of  oxidative  stress  

 
PURINE  SALVAGE:    recycle  and  reuse  for  purine  synthesis  when  cell  is  in  need  of  DNA  replication  
• What  cells  utilize  purine  salvage  pathways?  Actively  dividing  cells  
• What  are  the  exact  substrates  that  are  salvaged?  Free  purine  bases,  which  include  
o Hypoxanthine  …salvaged  to    IMP  
o Guanine  …salvaged  to    GMP  
o Adenine  …salvaged  to    AMP  
• What  are  the  purine  salvage  enzymes?  
 Hypoxanthine  &  guanine  use  HGPRT  (see  right)
o
 Notice  how  PRPP  is  used  here  (remember  this  guy  from  purine  &  pyrimidine  synthesis?)
o Adenine  uses  APRT  (adenine  phosphoribosyl  transferase)  
• What  are  sources  of  xanthine?    
o Adenosine  &  guanosine  nucleotides  
• What  disease  is  associated  with  lack  of  HGPRT?    Lesch-­‐Nyhan  syndrome  
o X-­‐linked  point  mutation  (one  single  nucleotide  difference!)  
o Results  in:  
 Hyperuricemia  Gout  
 Mental  retardation  
 Cerebral  palsy  
 Self  mutilation  
o HGPRT  deficiency      
 increased  turnover  of  purines  (since  salvage  is  blocked)    
 unused  PRPP  stimulates  increased  synthesis  of  purines  
o allopurinol  helps  reduce  the  hyperuricemia  &  gout  pain,  but  not  the  neurological  Sx  
 
PYRIMIDINE  CATABOLISM:    degradation  leads  to  NH3  (urea  excreted)  and  energy  
• What  intermediate  in  thymine  catabolism  is  proportional  to  the  amount  of  tissue  damage?    β-­‐aminoisobutyric  
acid  (AIBA)  
 
PYRIMIDINE  SALVAGE:    recycle  and  reuse  free  pyrimidine  bases  for  biosynthesis  
• What  two  steps  are  needed  to  go  from  free  base  to  usable  nucleotide  form?      
o Attachment  of  base  to  ribose  
o Phosphorylation  of  nucleoside  to  monosphophate  nucleotide  
• There  are  different  enzymes  with  specificity  for  various  pyrimidine  bases  to  add  the  ribose  back  on  
• Thymidine  kinase:    adds  phosphate  onto  thymidine  to  make  dTMP  (see  “pyrimidine  biosynthesis”  section)  
 
 
 

Happy studying!    

 
 
REFERENCES:  
Hubio514  lecture  notes  /  syllabus  

http://www.wikipedia.org    (when in doubt, wiki it out ;)

http://web.virginia.edu/Heidi/chapter27/chp27.htm  

http://seqcore.brcf.med.umich.edu/mcb500/nametov.html  (a really nice resource)