2. INTRODUCTION
A metabolic pathway is a series of chemical reactions in a cell
that build and breakdown molecules for cellular processes.
Metabolic pathway synthesis deals with the construction of
stoichiometrically consistent routes of biochemical (enzyme-
catalyzed) reactions that meet certain specifications.
Pathways are constructed recursively proceeding from the
substrate to the product.
Available substrates for the next round of expansion include
all products that the pathway has produced up to that point.
Reflects an approach that starts from a
given substrate and lists all possible
reactions that can deplete this
designated substrate.
3. Capacities for Participation: (Pathway Synthesis Algorithm)
I. as net reactants of the pathway, where there is net depletion of the
metabolite;
II. as net products of the pathway, where there is net production of the
metabolite;
III. as intermediates, in other words, participating without net consumption
or production.
Imposition of Constraints/Restrictions:
I. required reactants, whereby they must be consumed by the pathway
and their stoichiometric coefficient is strictly less than zero;
II. as allowed reactants, where they may or may not be consumed by
the pathway and their stoichiometric coefficient in the pathway is less
than or equal to zero;
III. excluded reactants (or prohibited reactants), which must not be
consumed by the pathway.
4. A CASE STUDY: LYSINE BIOSYNTHESIS
Synthesis of lysine from glucose and ammonia.
In order to simplify the case study, the enzyme α-ketoglutarate
dehydrogenase was assumed to be nonfunctional and was excluded
from the database.
glyoxylate shunt has been
invoked in order to
complement the TCA
cycle
and make up for the
absence of α-
ketoglutarate
dehydrogenase activity.
The
overall pathway
includes the basic units
of glycolysis, TCA
cycle, the
pathway from
oxaloacetate to
aspartate, and the
sequence of reactions
between aspartate and
lysine.
5. Exploring possibilities to bypass a single reaction if it turned out that
such a reaction constituted a bottleneck in the overall pathway. Eg- if we
assumed that malate dehydrogenase –’key limiting enzyme’ in the
pathway, it would be desirable to generate alternative pathways
bypassing this enzyme.This pathway bypasses the
whole TCA cycle through
the direct
carboxylation of pyruvate to
oxaloacetate, which can be
achieved by either
pyruvate carboxylase or
oxaloacetate
decarboxylase
This pathway also yields
a more attractive maximum molar yield.
If we neglect constraints from redox
and ATP balances, the maximum yield of
the pathway of is 100%
on a molar basis, compared to a molar
yield of only 67% for the former
pathway.
6. An alternative bypasses malate dehydrogenase with a set of just two
reactions converting malate to oxaloacetate:
i) malate + pyruvate oxaloacetate + lactate
Enzyme-lactate- malate transhydrogenase.
ii) lactate pyruvate
Enzyme- lactate dehydrogenase (in the reverse direction).
Another alternative, involves the conversion of malate to fumarate by
fumarase in the direction opposite that of previous one and conversion
of succinate to fumarate by succinate dehydrogenase as in the original
pathway.
.
Additionally, fumarate is converted
into aspartate through aspartate
amino lyase. Because
oxaloacetate is used in order to
form citrate, half of the aspartate
must be recycled into oxaloacetate
to close the TCA cycle.
The reaction of aspartate
glutamate transaminase
converts aspartate to
oxaloacetate by operating
in the reverse
direction of that of the
original bioreaction network.
7. ROLE OF OXALOACETATE
A key question – Can the metabolite be bypassed?
No single reaction surrounding oxaloacetate is fixed in the sense that it
is present in all pathways.
Particular reactions consuming and producing oxaloacetate may vary;
however, the intermediate itself is always present.
In the last pathway, aspartate and lysine are not derived directly from
oxaloacetate,
Reason: since fumarate is converted to aspartate by a single enzyme. In
fact, aspartate is converted into oxaloacetate, rather than the reverse.
Necessary TCA intermediates (malate or succinate) cannot be produced
from glucose without the intervention of oxaloacetate.
8. RESTRICTIONS ON THE MAXIMUMYIELD
No pathway was found that can produce lysine from glucose without
involving oxaloacetate as intermediate.
The maximum yield of the pathway can exceed 67% only through
carbon dioxide fixation by a carboxylation reaction.
Indeed, if carboxylation reactions are eliminated, the yield is
restricted to 67% or less.
Eg:- if a pathway were devised to convert 2 mol of pyruvate to 3 mol of
acetyl-CoA (without production or consumption of CO2), a yield of lysine
over glucose greater than 67% would be possible.
NOTE: However, within a reasonably complete database, no such
pathway exists.
Maximum yields are obtained with respect to carbon consumption
only.
9. REFERENCE
Mavrovouniotis, M. L. (1989). Computer-aided design of
biochemical pathways. Ph.D. Thesis, MIT, Cambridge, MA.
Mavrovouniotis, M. L., Stephanopoulos, G. & Stephanopoulos, G.
(1992a). Computer-aided synthesis of biochemical pathways.
Biotechnology and Bioengineering 36,
