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Specialty Chemistry Forums => Biochemistry and Chemical Biology Forum => Topic started by: batillia on November 28, 2012, 06:54:34 PM

Title: Wheat Germ Acid Phosphatase Activity?
Post by: batillia on November 28, 2012, 06:54:34 PM

Assume that wheat germ acid phosphatase (mw 55,000; pI=7.4) requires an aspartic acid residue (pK 4.5) and a histidine residue (pK 6.0) for activity. What is the percentage of maximal activity still present at pH 7.2?

The question says it requires a calculation, but I don't think I'm familiar with it... or maybe I'm totally missing it ^^;

Any help is appreciated!

Title: Re: Wheat Germ Acid Phosphatase Activity?
Post by: Borek on November 28, 2012, 07:00:42 PM

Looks to me like it is about fraction of the given form in the solution of a given pH. Acid dissociation equilibrium.

Title: Re: Wheat Germ Acid Phosphatase Activity?
Post by: Babcock_Hall on November 28, 2012, 07:21:52 PM

I suspect that one or assumptions will be needed.  One is that the Asp must be deprotonated (negatively charged), and the His must be protonated (positively charged).  Under these conditions, the graph of activity versus pH has a classic bell shape.  The second assumption is more subtle.  Do the two pK_a values refer to the free enzyme or to the E·S complex (they are generally not the same values)?  If we assume that they refer to the free enzyme, then it is possible to calculate the effect of pH on (V_max/K_m), which is the apparent first-order rate constant at very low substrate concentration.  Did your instructor provide any equations?

Title: Re: Wheat Germ Acid Phosphatase Activity?
Post by: batillia on November 29, 2012, 12:21:21 AM

He didn't give any equations...

We did a lab where we combined the WGAP with P-nitrophenylphosphate and buffers of different pH's, then we compared the resulting absorbance values to the buffer pH's. This was one of the added questions for us to answer.

Title: Re: Wheat Germ Acid Phosphatase Activity?
Post by: batillia on November 29, 2012, 06:46:47 PM

Sorry about the double post, but I'm really not understanding this question at all...

Anyone have any ideas..?

Title: Re: Wheat Germ Acid Phosphatase Activity?
Post by: Babcock_Hall on November 30, 2012, 09:15:58 AM

I will follow Voet and Voet's treatment in the third edition of Bichemistry (p. 361).    EH₂⁺ ::equil:: EH ::equil:: E^-.  The acid dissociation constant for the first dissociation is K_E1, and the acid dissociation constant for the second dissociation is K_E2.  V_max and K_m are the pH-independent parameters of the monoprotonated (active) form of the enzyme, EH.  The apparent values (which depend on pH) are V'_max= V_max/f2 and K'_m = K_m(f1/f2)  Therefore V'_max/K'_m = (V_max/K_m)(1/f1).  f1 =[H⁺]/K_E1 + 1 + K_E2/[H⁺]

It should be possible to calculate V'_max/K'_m from the pH and the two pK_a values you were given.

Title: Re: Wheat Germ Acid Phosphatase Activity?
Post by: batillia on November 30, 2012, 02:27:13 PM

I got it ^^ It was a lot simpler than I thought... but I guess since it's been so long since we had to use Henderson-Hasselbalch I didn't even consider it.

Thank you for your *delete me*