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Alkaline Phosphatase Kinetics

Alkaline Phosphatase Kinetics

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Published by Ina Keyser
To determine the kinetic parameters Vmax and Km of the non-allosteric enzyme, alkaline phosphatase, by investigating the optimum pH and temperature at which this enzyme functions.
To determine the kinetic parameters Vmax and Km of the non-allosteric enzyme, alkaline phosphatase, by investigating the optimum pH and temperature at which this enzyme functions.

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Categories:Types, Research, Science
Published by: Ina Keyser on Nov 09, 2012
Copyright:Attribution Non-commercial

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04/21/2014

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BCM 254 & BCM 256
Alkaline Phosphatase Kinetics
Ina Keyser 10144383
University of Pretoria14 May 2011
 
2
INDEX1.
 
INTRODUCTION 3a)
 
Aim 42.
 
METHOD 5a)
 
Materials and Reagents 5b)
 
Procedure
i.
 
Determining the Optimal pH 5ii.
 
Determining the Optimal Temperature 6iii.
 
Determining the Vmax and Km Values 6
3.
 
RESULTS 7a)
 
Optimum pH 7b)
 
Optimum Temperature 8c)
 
Optimum Substrate Concentration 10d)
 
Calculation of Vmax and Km Values 114.
 
DISCUSSION 13a)
 
Optimum pH 13b)
 
Optimum Temperature 13c)
 
Optimum Substrate Concentration 145.
 
CONCLUTION 146.
 
REFERENCES 16
 
3
1.
 
INTRODUCTION
Enzymes make up a large and diverse group of (mainly) proteins that are essentialfor metabolic functions in cells. These substrate-specific molecules function ascatalysts in virtually all biochemical reactions by decreasing the activation energyrequired for the conversion of the substrates (or reagents) to products.Enzymes are usually composed from amino-acids with R-groups, each with definingcharacteristics. The degree to which the R-groups of amino acids are protonated at aspecific pH, determines the charge on those amino acids. And when present in anenzyme, the level of protonation of the various R-groups determines the degree of protonation and, therefore, the charge of the enzyme as a whole. The level of protonation of the individual R-groups, however, are responsible for the number andtypes of electrostatic interactions and hydrogen bonding; and in turn the three-dimensional structure of the enzyme. Should the pH of the existing environmentchange, the protonation of the R-groups will vary and the electrostatic interactionsand hydrogen bonding will be redistributed so that the enzyme conformationchanges as a whole. Not all the enzymes in the population undergo these changes inconformation. This result in either an increase or decrease in the number(concentration) of enzyme molecules present in the active conformation and can beused as a measure for the rate of the reaction.For many enzymes the rate of catalysis, defined as the number of moles (n) of product formed per second (s), varies with the substrate (S) concentration. Theextent of product (P) formation is determined as a function of time for a series of substrate concentrations. As expected in each case, the amount of product increaseswith time, although eventually a time is reached when there is no net change in theconcentrations of S or P. The enzyme is still actively converting S into P and vice-versa, but the reaction has attained a dynamic equilibrium. [1]The reaction to be investigated is that catalysed by alkaline phosphatase (AP).Alkaline phosphase is a hydrolase present in all tissues throughout the human body,but particularly concentrated in the liver, bile duct, kidneys, bone and in pregnantfemales
 –
the placenta. It is responsible for the hydrolysis of phosphor-ester bonds,releasing a phosphate group and the alcohol derivative of the substrate. [1, 3]Alkaline phosphase
needs Mg²⁺ ions to function and is inhibited by chelating agents
(e.g. EDTA) and inorganic phosphate. In this experiment p-nitro phenol-phosphatewill be used as a substrate for the synthesis of p-nitro phenol. P-Nitro phenol isyellow and p-nitro phenol-phosphate is colourless. It is therefore easy to perform aspectrophotometric enzyme assay.

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