Metabolic
regulation of heme biosynthesis.
Piotr Pawlowski, Marta Hoffman, Monika Gora, Joanna Rytka, Piotr Zielenkiewicz
Institute of Biochemistry and Biophysics, Polish Academy of Sciences,
Pawinskiego 5a, 02-106 Warsaw, Poland (martah@ibb.waw.pl)
We developed a
theoretical model of a chain of irreversible Michaelis-Menten reactions,
proceeding inside a living cell, taking cell growth and division and
subcellular compartmentation into account. We also constructed an algorithm for
enzymatic data modeling - a tool allowing the estimation of lacking parameter
values for the modeled system, in accordance both with the canons of the
proposed theoretical model and the available experimental data. The model was
then applied to the special case of heme biosynthesis in the yeast Saccharomyces
cerevisiae,
in an approach to identify the rate-limiting steps of this pathway. All
required experimental values were obtained either from literature, or by means
of the proposed algorithm. The resulting model was validated with data on the
pathway's behavior under modified environmental conditions and in mutant yeast
strains, and was then used for various simulations. These allowed the
identification of four enzymes as rate-limiting: porphobilinogen synthase,
porphobilinogen deaminase, uroporphyrinogen III decarboxylase and
coproporphyrinogen III oxidase. Here we present partial experimental
verification of these results. By constructing yeast strains which overproduce
two enzymes - the porphobilinogen synthase and deaminase - and characterizing
them biochemically, we show that the pattern of intermediate accumulation in
the engineered cells confirms the rate-limiting role of both enzymes.