The Next 4 Slides Are Background Material (Review of Previous Lectures)

M1 – Biochemistry

Nucleotide Synthesis and Degradation I

Dr. Ratz

(Resources: Lehninger et al., 5th ed., Ch 22, pp 882-896)

THE NEXT 4 SLIDES ARE BACKGROUND MATERIAL (REVIEW OF PREVIOUS LECTURES)

2 REVIEW Slide #1 The sugar – 5-C’s: RECALL Nucleotide Components: 1’ 1. Phosphate(s) 2’ 2. Sugar (Ribose or 3’ 4’ Deoxyribose) 5’ 3. Nitrogenous base To be a These are “D” enantiomers: nucleotide, these (chiral C-4 –OH is to the right Figure 7.1(c) Lehninger 4th ed. –OH’s are linked in this Fisher projection) to a Pi & base

H+ Glycosyl (or glycosidic) bond) 1’ 2’ 5’ β When –OH is up 4’ 1’ 3’ α When –OH is down .. 2’ 4’ 3’ 5’ 1 5 O 2 1 2 C C (resembles furan) 3 α-Furanose 4 C C 3 Furan Recall that ribose can hemiacetals (tetrahydro-, exist as 3 isomers: (no longer an aldehyde) in this case)3 Ch 8 Lehninger 5th ed.

REVIEW Slide #2 RECALL Nucleic 1. Phosphate(s) Acid structure: 2. Sugar 3. Nitrogenous base Nucleotide: (nucleoside PLUS 5’ Pi(‘s))

Nitrogenous Base H+ H+ glycosyl bond

- Note numbering of sugar (pentose) is CW. - Note the primes used to # the pentose (sugar in nucleic acids is #’d this way). Ch 8 Lehninger 5th ed. 4 REVIEW Slide #3 Common nitrogenous base structures & names

1 6 2 9

4 5 2 1

Last REVIEW Slide (#4) Review of nomenclature

ψ ∗

(())(RNA)

Ribose-Base Pi(s)-Ribose-Base Polymer

Difficult name to remember (cytosine suggests it’s a nucleoside, but cytidine is the nucleoside name) ψ (e.g. of nomenclature: ATP, a ∗ 1. Adenylate = AMP = adenosine 5’ monophosphate = adenylic acid nucleotide, is called adenosine 5’ 6 triphosphate) 2. Note that AMP, ADP & ATP are nucleotides Nucleotide Synthesis and Degradation I

Learning Objectives: 1. Recognize the importance of nucleotides in biosynthetic and other biochemical reactions. 2. Know the precursors of the purine and pyrimidine ring structures. 3. Have general knowledge of the de novo synthetic pathways and their control mechanisms.

Nucleotide Synthesis and Degradation I

Importance of Nucleotide Biosynthesis: - Nucleotides (especially the nucleotide ATP (a nucleoside triphosphate), & to a lesser extent, GTP) serve as carriers and reservoirs of chemical energy that help drive almost all biosynthetic events. Some examples: DNA & RNA Proteins Complex polysaccharides Glycolipids & Phospholipids

- ATP is the most abundant nucleotide in the cell because of its central role in these numerous, coupled, anabolic reactions.

- The ribo- and deoxyribonucleoside triphosphates are the precursors of RNA and DNA synthesis. If the levels of these nucleoside triphosphates are not maintained in sufficient quantity, particularly during hyperplastic growth, then RNA & DNA synthesis can be rate limited by precursor availability.

8 Importance of Nucleotide Biosynthesis: -Nucleotides are also components of cofactors, such as: NAD (hydride ion (:H- or H+ + 2e-) transfer) FAD (hydrogen atom (:H or H+ + e-) transfer)

S-adenosylmethionine (methyl group transfer) (-CH3) CoA (Coenzyme A) (acyl group transfer) -C=O Acetyl,

CH3 for eg -Nucleotides are components of activated biosynthetic intermediates, such as: UDP-glucose (glycogen synthase; Fig 15.8 & 15.11, Lehninger, 4th ed) CDP-diacylglycerol (phospholipid head group synthesis; Fig 21.24)

-Nucleotides are components of intercellular communication, such as: ATP released by certain nerve terminals, and its metabolite, adenosine, bind to specific postsynaptic purinergic receptors

-Nucleotides are components of intracellular communication (cellular messengers), such as: cAMP cGMP 9

Nucleotide Synthesis and Degradation I Learning Objectives: 1. Recognize the importance of nucleotides in √ biosynthetic and other biochemical reactions. 2. Know the precursors of the purine and pyrimidine ring structures. 3. Have general knowledge of the de novo synthetic pathways and their control mechanisms.

10 There are 2 types of pathways leading to nucleotide synthesis: 1. De novo synthesis: Nearly identical in all living organisms. Synthesis begins with their metabolic precursors- i. Ribose 5-phosphate Nitrogenous ii. Certain amino acids Base

iii. CO2 iv. NH3

2. Salvage pathways: Recycle the 2 major components released from nucleic acid breakdown- Nucleosides Free Bases

1. De novo nucleotide synthesis metabolic precursors: i. Ribose 5-phosphate (Where does this come from?) ii. Certain amino acids (Where do these come from?)

iii. CO2 (directly in the form of bicarbonate!) iv. NH3 (released from glutamine)

12 De novo synthesis of purines

Fig 22.32, Lehninger, 5th. Origin of the ring atoms of purine (4 N’s, 5C’s):

(amino Glycine 5th (amino 2nd nitrogen) nitrogen) th 6 1 6 5 7 8 2 4 rd 7th 3 9 3 1 C transfer using N10-formyl- th 4 1st tetrahydrofolate (see Fig 18.17) – aldehyde transfer +

subsequent loss of H2O. Most commonly, formate C is, itself, donated from serine-to-glycine Let’s start here (amido conversion in synthesis nitrogen) (ultimately) of N10-formyl-THF. 13

Construction of purine nucleotides The purine bases are constructed, one or several atoms per step, upon the substrate, 5-phosphoribosyl-1-pyrophosphate (PRPP) 5 N- You saw this compound before, in the 4 1 synthesis of histidine & tryptophan. 3 2 PRPP is synthesized from ribose 5- phosphate derived from the pentose phosphate pathway + ATP p 861, Lehninger 5th ed.

Ribose 5-Pi + ATP Æ 5-PRPP + AMP

Ribose phosphate pyrophosphokinase (highly allosterically regulated)

14 The purine bases are constructed, one or several atoms per step, upon the substrate, 5-phosphoribosyl-1-pyrophosphate (PRPP) 5 :NHN-2- PRPP plays a role in de 4 1 3 2 novo biosynthesis of both purines and pyrimidines

p 861, Lehninger 5th ed.

- As in the case with histidine and tryptophan synthesis, C-1 of PRPP undergoes a nucleophilic attack by electrons of a nitrogen, releasing pyrophosphate - As opposed to histidine and tryptophan synthesis, the ribose ring structure is retained in the nucleotide.

SUMMARY: The sugar component of purines is ultimately derived from glucose, and proximally derived from 5- phosphoribosyl- 1-pyrophosphate ATP AMP PRPP Ribose phosphate pyrophosphokinase

16 Pentose Phosphate Pathway. From Fig 14.20, Lehninger 5th ed. The 1st committed step in purine synthesis is the transfer of the amide (amido) N from glutamine to PRPP as shown in the following reaction:

(PRPP)

5-Phospho-β- D-ribosylamine

Gln17 From Fig 22.33, Lehninger 5th ed.

The 2nd step in purine 1 step (6a) in synthesis is the higher eukaryotes addition of 2 carbons & 1 N from glycine:

5-phospho-β- D-ribosylamine

The remaining steps: DON’T MEMORIZE! As in histidine & tryptophan biosynthesis, enzymes in this pathway are arranged as multienzyme complexes. This is th Fig 22.33, Lehninger, 5 ed. a nucleotide De novo synthesis of purine nucleotides – construction Inosinate 18 of the purine ring, inosinate (IMP) Remember this slide from an earlier lecture?

An aside – What methylated purine bases are found in plants that are harvested for consumption in beverages?

5 6 1 theobromine 4 3 2 caffeine purine xanthine theophylline (nucleoside) inosine hypoxanthine (nucleotide) inosinate (No C-2 =O) The nucleotide, inosinate (I), containing the uncommon base, hypoxanthine, is found in some anticodons

Fig 22.34, Lehninger, 5th ed. De novo synthesis of purine nucleotides – conversion of inosinate to adenylate and guanylate.

(Ahh – TCA intermediates Similar to step all over again!) Note that (8), but uses GTP H synthesis of rather than ATP AMP requires GTP, and synthesis of GMP requires ATP! Hence, these two pathways can help regulate (and BALANCE) each other by the relative concentrations of ATP and GTP in the cell.

(oxidation; then amido

(–NH2) is “exchanged” Similar to step (4) for =O)

th Fig 22.34, Lehninger, 5 ed. 20 Biosynthesis of AMP and GMP from IMP The conversion of GMP and AMP to triphosphates to be used in biosynthesis is carried out by

1. kinases, and 2. oxidative phosphorylation (OXPHOS): * Base-specific nucleoside monophosphate kinases Guanylate Kinase* (guanylate kinase here; see GMP + ATP ÅÆ GDP + ADP adenylate kinase below) Oxphos 2 ATP Kinase** ** Ubiquitous nucleoside GDP + ATP ÅÆ GTP + ADP diphosphate kinase has broad specificity (phosphate is “donated” usually from ATP to any NDP – & dNDP) Adenylate Kinase* Oxphos AMP + ATP ÅÆ 2ADP Æ 2ATP

We have now made the purine precursors for ribonucleic acid biosynthesis21

Nucleotide Synthesis and Degradation I Learning Objectives: 1. Recognize the importance of nucleotides in √ biosynthetic and other biochemical reactions. √ 2. Know the precursors of the purine and pyrimidine ring structures. √ purines 3. Have general knowledge of the de novo synthetic pathways and their control mechanisms.

22 Formation of 5-phospho-β-D-ribosylamine has a single fate in metabolism. Thus, the committed step is highly regulated by feedback inhibition of the overall process.

The regulatory Mechanisms for …and by other biomolecules Purine Nucleotide Synthesis – PRPP

The most important (i.e., 1st level) of these is feedback inhibition of the committed step, PRPP to 5- phosphoribosylamine

2nd level of regulation: Short feedback 3rd level of regulation - loops “balancing” ATP & GTP production: use of GTP vs ATP for synthesis of adenylate & guanylate from inosinate (previous slide)

Fig 22.35, Lehninger, 5th ed. Regulatory mechanisms (E. Coli) – 23 regulation differs in different organisms.