[Natasha]

I am trying to convert the value of Vmax to kcat given in this reference: https://www.ncbi.nlm.nih.gov/pubmed/7488210

Vmax value of 1.7nmol/mg/min is given for phosphofructokinase.(in page 901 of the ref.)

When Vmax is multiplied with the molecular weight(82000 g/mol),the resulting value of kcat is in the order of 10^(-9) (units: 1/s)which is too low.

Am I missing something?

[Babcock_Hall]

I only have time for a quick thought right now, but I might be able to return to this subject later on.  PFK-1 is a tetramer of four identical subunits.  IIRC k_cat can be calculated either on the basis of the number enzyme molecules or the number of active sites, and offhand I cannot recall which is preferred.  This would only potentially account for a fourfold difference, however.

[Yggdrasil]

When I do the calculation, I get something on the order of 10^-4 s^-1.  Double check your calculation, paying special attention to the units (e.g. need to convert between nmol and mol, mg and g, min and s).

[Natasha]

Thanks a lot for the calculation.Just to be sure of the steps,here is another example

The value of Vmax reported in the supporting information(http://journals.plos.org/ploscompbiol/article/file?id=10.1371/journal.pcbi.1002577.s013&type=supplementary) of http://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1002577#s5  is 7.182 micro mol/kg/min for the same enzyme phospho fructo kinase.

Molecular weight reported in uniprot is 85018 g/mol

The kcat value which I obtain is 1.0176 x10^-5 (1/s)

In brenda ,a turn over number of 357 is reported for http://www.brenda-enzymes.info/enzyme.php?ecno=2.7.1.11&Suchword=&reference=&UniProtAcc=&organism%5B%5D=Homo+sapiens&show_tm=0
Any suggestions on why this difference?

[Natasha]

Could you please tell where one can find information on PFK-1 being a tetramer?

[Babcock_Hall]

Figure 15-16 in Principles of Biochemistry, 6th ed., by Nelson and Cox show E. coli PFK-1 (PDB ID 1PFK) as a tetramer, and other textbooks give similar figures.  Obviously, other organisms could conceivably have different quaternary structures, and one should check, to be certain.  But this will only potentially affect the value by a factor of 4.  Also, different organisms may have different molecular weights of the subunits, but this will also have a relatively small effect on the calculation.  I think that there might be a problem with your conversions of units.

[Babcock_Hall]

In the 2nd edition of Biochemical Calculations, Segel indicated (pp. 282-283) that turnover number can be used in two ways.  Whem the number of moles of substrate transformed per mole of enzyme is calculated, this is can be called the "molecular activity." When the number of moles of substrate per moles of catalytic center is calculated, this is called "catalytic center activity."  I am not sure how universal these terms are.  On page 286 Segel gave a problem with solution for calculating the turnover number.

Why not write out your calculation for us to look at?

[Yggdrasil]

From the PLoS Comp Bio reference, I am not sure that the V_max used by the authors corresponds to the standard definition of V_max in biochemistry.  I think the number they report is basically just the PFK1 activity per kilogram of liver tissue.  To find the turnover number, you would need to know the amount of enzyme present in one kilogram of liver tissue.

[Natasha]

Sorry for the delay in my response.
If I understand correctly the units of Vmax in the  PLoS Comp Bio reference is given in micromoles/min/kg body weight.I didn't pay attention to this information before,which is why the kcat  that I calculated was too low.
I believe,in vitro Vmax has been converted to in vivo Vmax.(ref:New technologies for toxicity testing / edited by Michael Balls, Robert D. Combes, Nirmala Bhogal.)

Vmax (in vivo) = Vmax (in vitro) x Cprot x Ftiss

Vmax (in vivo) is maximal velocity of metabolism in vivo (mg/min per kg body weight),
Vmax (in vitro) is maximal velocity of metabolism in vitro (mg/min/mg microsomal protein),
Cprot is concentration of microsomal protein (mg/g tissue) and
Ftiss refers to the fractional volume of the metabolizing tissue (e.g., g liver/kg body weight)

From the in vivo Vmax given in the PLoS Comp Bio reference,I would like to calculate the in vitro Vmax for which I need the values of quantities Cprot and Ftiss.
In page 250,of the ref:https://www.crcpress.com/Principles-and-Methods-of-Toxicology-Fifth-Edition/Hayes/p/book/9781420005424

The Tissue volume (fraction of BW) for liver is given as 0.026.Where ,BW is Body weight of human: 70kgs
Would it be appropriate to use this value for Ftiss in the above equation.

Any suggestions on how to find the Cprot value for a given protein?I wish to obtain the Cprot values for all the enzymes present in glycolysis pathway.

I would be grateful for any help you can offer.

[Yggdrasil]

You could try searching the BioNumbers database (http://bionumbers.hms.harvard.edu/) for some of the values, though I'm not sure you would be able to easily find the Cprot values.  Cprot values may also differ between individuals and within the same individual over time, so it's unclear whether you could get a reliable estimate from combining numbers from different sources.

[Babcock_Hall]

I would also look into the literature on metabolic control analysis, which is sometimes used to study the flux through pathways.  Figure 15-9 in Nelson and Cox, Principles of Biochemistry, 6th ed. covers hexokinase IV, PFK-1, and PHI from the point of view of which one controls the flux in the first portion of glycolysis in the liver.  This is done by adding purified amounts of each enzyme to an extract.  Unfortunately, it is unclear what the journal reference is for this figure.  I am not sure whether or not the authors of the original study found the amounts of each enzyme in vivo.  However, my best guess is de la Iglesia et al., (2000) J Biol Chem 10:597-603.