calculate the mole of substrate in assay

[scholesmu]

"alkaline phosphate is assayed at concentration 1ug/ml at r.t. in 0.1M glycine buffer, pH9.5 in presence of 5mM MgCl2 as a factor in reaction. The substrate 4-nitrophenol (4-NPP) is added in concentration range 0.05 mM to 3.0mM. The appearance of the product, 4-nitrophenol (4-NP) can be moitored at 420nM (E=15mM-1cm-1)

Vmax has been estimated to be 0.1umol/min/assay
Km is 0.15mM under conditons described in the legend.
The MW of enzyme is 100000

If i want to calucalate
1) If the assay was performed at a substrate concentration 3mM how many nmole of substrate would 1ug of alkaline phosphate converted to product in 1min?

2) If the assay was performed at a substrate concentration 3mM how many mmole of product would 1mmole of enzyme release in 1min?


these 2 questions i am stuggling...
answer 1) is 33.3 and 2) 3333

[JackBean]

it's alkaline phosphatASE

[scholesmu]

thanks...
do you know how to calculate? i am stuggling...

[JackBean]

well, the concentration is 20× higher than K_M, so I would assume that the reaction procceds with speed of V_lim, that is 0.1 umol/min/1(ug/ml) what should make it 100 nmol for 1) :-/