Problem 3. Enzyme kinetics and Human milk is Nature’s first and probably best functional food. The…

Problem 3. Enzyme kinetics and
Human milk is Nature’s first and probably best functional food.
The third most abundant component of mother’ milk comprises more
than 100 sugar oligomers collectively referred to as human milk
oligosaccharides (HMO) that select for beneficial bacteria and
jump-start the gut microbiota of the infant. HMOs are assembled by
adding β-(1→3)-linked repeating units of the disaccharide LNB
(lacto-N-biose: Galactose-β-(1→3)-N-acetyl
Glucosamine) to a lactose (Galactose-β-(1→4)-Glucose) at the
reducing end. This motif can be fucosylated. e.g. the
Lacto-N-difucohexaose I (LNDFI) shown below. A research
group has recently discovered a new enzyme that is able to degrade
LNDFI to lactose and to the Lewis b tetraose as shown below:

The kinetics of hydrolysis of this enzyme towards LNDFI were
analysed by a coupled assay measuring the concentration of the
lactose product. The initial rates are reported in Table 1.

D) Determine kcat if [Enzyme F] = 20 nM
Lacto-N-difucohexaose (LNDFI) Lewis b tetraose Lactose Enzyme F – + Galactose (Gal) Fucose (Fuc) 5 3 2 Glucose (Glc) IN-acetyl glucosamine (GlcNAc) Figure 1. The hydrolysis of Lacto-N-difucohexagse I to LNB and lactose by the enzyme F. The key shows the monosaccharide composition and the linkages between the monosaccharides (e.g. 4 means that there is a 1-4 glycosidic linkage and all linkages are in the D-B configuration, except for fucosyl that is linked via a-L- linkages. The glycosidis bond that is cleaved is the D-B-(1.3)-bond between the GlcNAc and the Gal. Table 1. Hydrolysis kinetics of enzyme F towards LNDFI LNDFI (mm) V. (UM/s) 0.10 0.15 0.25 0.40 0.50 0.60 1.00 1.50 2.50 *Structure of Lewis a triose: 0.186 0.281 0.409 0.580 0.728 0.755 0.899 1.040 1.300 V. in presence of 25 mm added Le a triose* (M/S) 0.072 0.105 0.169 0.256 0.309 0.358 0.526 0.687 0.911