Chem*4520 Metabolic Processes Fall Semester 2000 Modified November 2000
schematic view of the enzyme citrate synthase, with bound acetyl-CoA analog in green	Department of Chemistry and Biochemistry Home Page Back to Chem*4520 Home Page

Solutions to problem set 8, Nov 14-20

1.

This is achieved by use of highly activated phosphorylated precursors, including 2 moles of PEP, and this represents a hidden energy cost on metabolism. The three major bond formation reactions use the existing phosphates on the initial substrates so external ATP is only needed for a single elimination step.

The use of transaminase as N donor is also a hidden energy cost, since it makes one glutamate unavailable for glutamate dehydrogenase, and one less NADH is produced. Amino acids are at the oxidation level of hydroxy acids, so conversion of hydroxyphenylpyruvate to tyrosine is a reduction step with a hidden cost of one NADH.

Which reactions are energetically favourable?

DAHP synthase		This reaction changes bonding in three ways: 1) loss of the high energy PEP phosphate should be favourable 2) conversion of enol to keto should be favourable 3) The C-C bond formation is an aldol type reaction. Comparable aldol reactions include citrate synthase ( = 0 for the bond formation step), malate synthase (= -14.8 kJ/mol for bond formation step), aldolase ( = - 22 kJ / mol in the gluconeogenesis or bond formation direction. Isocitrate lyase is a retro aldol type bond breakage (+8.7 kJ/mol). A ballpark estimate would predict that the aldol bond formation should be slightly favourable also. Since all three contributions appear to be favourable, the reaction as a whole should be highly favourable. The release of Pi as a second product also ensures that this is a two substrate - two product reaction, so there is no adverse effect on from low overall concentrations.
DHQ synthase		1) There's a phosphate elimination, although not a high energy phosphate like PEP. 2) The ring closure reaction is another aldol condensation. Both these components of the reaction should be favourable. In addition, this reaction has one substrate yielding 2 products (Pi is released), so is more negative at low overall concentrations.
DHQ dehydratase		Hydrations tend to be slightly favourablee.g. fumarase, = -3.8 kJ/mol, and dehydration are slightly unfavourable, e.g. enolase, = +3.2 kJ/mol. In this case conjugation of the double bond with the keto group may help, and we don't know how cyclohexane ring geometry will affect the reaction, but it's likely to be slightly unfavourable.
Shikimate dehydrogenase		This is a reduction of a ketone to a secondary alcohol by NADH (dehydrogenase in reverse). Secondary alcohol oxidations by NAD+ are not usually very favourable, ranging from hydroxyacyl-CoA dehydrogenase, = +15.7 kJ/mol to malate dehydrogenase = +29.7 kJ/mol. It's probably safe to assume that a ketone reduction by NADH is favourable.
Shikimate kinase		There's nothing to suggest that shikimate-5-phosphate is a high energy phosphate (enol phosphate, acyl phosphate) so phosphate transfer with ATP as donor is likely to be favourable.
Enolpyruvylshikimate synthase		This is a group transfer with PEP as donor. The enol phosphate is displaced, but the pyruvate component remains in the less stable enol tautomer form, so only half the energy of PEP is released. Nevertheless this should be enough to make the formation of the ether bond favourable.
Chorismate synthase		Elimination of phosphate introduces a double bond. Phosphate elimination can be considered the sum of phosphate ester hydrolysis (about -15 kJ/mol for an ordinary phosphate ester) and dehydration (slightly disfavoured), so the process is favourable overall. Unlike dehydration, where the second product is H2O, here Pi is a second product from a single reactant, making more negative at low overall concentrations.
Chorismate mutase		The pyruvyl group changes from the enol to the more stable keto form, so my best guess is that this is a favourable reaction.
Prephenate dehydrogenase		The decarboxylation plus the aromatization of the ring make a huge favourable contribution. Against this there is the hydride transfer to NAD+, but there are no simple analogous reactions for comparsion. A best guess would say that the favourable contributions are overwhelming.
Transaminase		Since the overall reaction consists of N- transfer from amino acid to PLP followed by almost the exact opposite process from PMP back to amino acid, typically transaminases have an equilibrium constant close to 1, and the reaction is neither favourable nor unfavourable.

2

Regulation of the initial reaction, DAHP synthase controls entry of substrate to the pathway.

Regulation of the branch point at chorismate controls the final product.

In E coli, both allosteric and gene repression mechanisms control activity.

Three DAHP synthases are expressed; each is under negative allosteric regulation by one of the aromatic amino acids. Each gene is repressed by the same amino acid, except that the Phe sensitive enzyme is repressed by both Trp and Phe. The branch point is controlled by the three enzymes:

anthranilate synthase (Trp, PABA, ubiquinone pathways)		negative allosteric effect of trp, repression by trp
chorismate mutase/prephenate dehydratase (phe pathway)		negative allosteric effect of phe, repression by phe
chorismate mutase/prephenate dehydrogenase (tyr pathway)		negative allosteric effect of tyr, repression by phe and tyr

Although prephenate is the true branchpoint, chorismate mutase exists in two forms, complexed with prephenate dehydrogenase or prephenate dehydratase respectively. Prephenate is never released as a product, so regulation must control the chorismate mutase as well. Chorismate is the last common intermediate released to the surroundings.

The tyrosine branch is regulated by phe as well, since tyrosine can also be obtained by phenylalanine hydroxylase.

3.

Ru-5-P: ribulose-5-P		epimerases and isomerases interconvert the pentoses
Xu-5-P: xylulose-5-P		TK: transketolase, transfers a 2-carbon unit, used again later
R-5-P: ribose-5-P
S-7-P: sedoheptulose-7-P		TA: transaldolase, transfers a 3-carbon unit
G-3-P: glyceraldehyde-3-P		output to glycolysis
Fru-6-P: fructose-6-P		output to glycolysis

When erythrose-4-P is an end product, a simplified pathway can operate:

The pathway can operate in reverse to provide ribose-5-P when no NADPH is needed:

If the oxidative pathway is modified by running the last TK reaction in reverse, 2 E-4-P are produced per mole of glucose-6-P oxidized and for one mole of G-3-P enetering from the right. This would represent the maximum efficiency of conversion to E-4-P

4.

Decarboxylation requires electron shift away from the carboxylate. The electron sinking action of PLP weakens bonds on carbons in odd number positions from the the Schiff N. This allows decarboxylation of the alpha-carboxylate but not decarboxylation of the beta carboxylate. The gamma carboxylate of glu is not susceptible, because the connecting carbon chain is not double bonded so does not extend the electon shift to gamma carboxylate of glutamate.

In contrast, elimination of OH^- requires electron shift towards the O. Return of electrons from the PLP sink can allow elimination of OH^- from the even carbon position in serine dehydratase.

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