The Kinetic of Alkali Phosphatase
Biochemistry Unit
The Kinetics of Alkali Phosphatase Inhibition
1. OVERVIEW
This practical builds on the enzymology lab skills you learned in the Acid Phosphatase practical.
Again, you will measure the initial reaction velocity (V 0) of an enzyme reaction, but this time in the absence and then presence of an inhibitor.
Last time you used Acid Phosphatase (Prac 1), but this time you will use the enzyme
Alkali Phosphatase. These enzymes have different primary (and hence tertiary) structures, pH optima and cofactors, so are more unlike than alike. However, Alkali Phosphatase can also catalyse the hydrolysis of the artificial substrate PNPP to PNP + P i, so PNPP will again be the substrate in this prac.
The inhibitor you will test will be the yellow-coloured product PNP – so this is an experiment to investigate product inhibition. What do you think will happen to V0 if the [PNP] were significantly increased? Again, you will be marked on the quality of your data and understanding of the experimental details. This prac also provides some nice enzyme inhibition data for you to use in later workshops.
Please observe meticulous good lab practice: no eating or drinking; wear lab coats at all times; handle chemicals carefully and with gloves; read, understand and think about what you are doing; cooperate as a pair and use common sense.
2. INTRODUCTION
The enzyme you will use is bovine Alkali Phosphatase (EC 3.1.3.1), bought from Sigma
Life Sciences (www.sigmaaldrich.com). As the name suggests, it has a pH optima above 7.0, requires divalent Mg2+ ions for activity, and can catalyse the hydrolysis of PNPP to p-nitrophenol
(PNP) and inorganic phosphate (see prac 1 schedule).
Remember that PNP is yellow at alkaline pH, and its appearance can be detected at 405 nm using a spectrophotometer. Because this enzyme works best at alkali pH, we do not need to use a timepoint-quench method (as we did with Acid P), but can monitor the ΔA 405 directly in
real
The Kinetics of Alkali Phosphatase Inhibition
1. OVERVIEW
This practical builds on the enzymology lab skills you learned in the Acid Phosphatase practical.
Again, you will measure the initial reaction velocity (V 0) of an enzyme reaction, but this time in the absence and then presence of an inhibitor.
Last time you used Acid Phosphatase (Prac 1), but this time you will use the enzyme
Alkali Phosphatase. These enzymes have different primary (and hence tertiary) structures, pH optima and cofactors, so are more unlike than alike. However, Alkali Phosphatase can also catalyse the hydrolysis of the artificial substrate PNPP to PNP + P i, so PNPP will again be the substrate in this prac.
The inhibitor you will test will be the yellow-coloured product PNP – so this is an experiment to investigate product inhibition. What do you think will happen to V0 if the [PNP] were significantly increased? Again, you will be marked on the quality of your data and understanding of the experimental details. This prac also provides some nice enzyme inhibition data for you to use in later workshops.
Please observe meticulous good lab practice: no eating or drinking; wear lab coats at all times; handle chemicals carefully and with gloves; read, understand and think about what you are doing; cooperate as a pair and use common sense.
2. INTRODUCTION
The enzyme you will use is bovine Alkali Phosphatase (EC 3.1.3.1), bought from Sigma
Life Sciences (www.sigmaaldrich.com). As the name suggests, it has a pH optima above 7.0, requires divalent Mg2+ ions for activity, and can catalyse the hydrolysis of PNPP to p-nitrophenol
(PNP) and inorganic phosphate (see prac 1 schedule).
Remember that PNP is yellow at alkaline pH, and its appearance can be detected at 405 nm using a spectrophotometer. Because this enzyme works best at alkali pH, we do not need to use a timepoint-quench method (as we did with Acid P), but can monitor the ΔA 405 directly in
real