Lab Activity4 IUG, 2015 Dr. Tarek M. Zaida

Enzyme Kinetics

Enzymes Enzyme reaction: E + S ↔ ES → E + P Whereas: E: Enzyme, S: Substrate, ES: Enzyme-Substrate complex, P: Product

Enzymes decrease activation energy Biochem 3070 - Exploring Genes Edward Walker 7/19/2019 Enzymes decrease activation energy A chemical reaction goes through a transition state with a higher G than either S of P Enzymes facilitate the formation of the transition state by decreasing G‡

Enzyme Kinetics Factors influencing an enzymatic reaction Substrate concentration Temperature Inhibitors Activators pH

1. Substrate Concentrate At low values of [S], the initial velocity,Vi, rises almost linearly with increasing [S]. But as [S] increases, the gains in Vi level off (forming a rectangular hyperbola). The asymptote represents the maximum velocity of the reaction, designated Vmax

The substrate concentration that produces a Vi that is one-half of Vmax is designated the Michaelis-Menten constant, Km is (roughly) an inverse measure of the affinity or strength of binding between the enzyme and its substrate. The lower the Km, the greater the affinity (so the lower the concentration of substrate needed to achieve a given rate).

Allosteric effectors Noncovalently bind and regulate the enzyme. The effector may be stimulatory or inhibitory. The substrate and effector usually occupy different specific binding sites.

Enzyme Kinetics of Alkaline Phosphatase (ALP)

Background substrate product product Function R-PO4 + H2O → R-OH + H3PO4 substrate product product The reaction catalyzed by alkaline Phosphatase

Experiment Facts.. An assay is necessary to study an enzyme The assay is a measurement of a chemical reaction, which might involve measuring the formation of the product (or otherwise the decrease in substrate conc.)

Reagents and instruments 18 labeled plastic tubes Micropipette Spectrophotometer ALP enzyme kit (ready to use) Blood serum 5 N NaOH solution

Procedure Take 18 clean plastic tubes and label them from 1 to 18. Another tube will be used as a blank. This will contain all the reagents except of the enzyme. Make the substrate and buffer concentrations as described in the given table (make sure to keep the total volume of all tubes stable at 1.9 ml). Transfer 100 µl of serum to each tube. Mix substrate and serum solutions and incubate at 37c for 50 seconds. Add 0.5 ml of 5 N NaOH in each tube to stop the reaction. Read the absorbance at 400 nm. Plot reaction rate (Vi) on Y axis against substrate concentration [S] on X axis.

Abs. at 400nm Tube Substrate μl Buffer Enzyme 5 N NaOH 1 100 1800 500 2 200 1700 3 300 1600 4 400 1500 5 1400 6 600 1300 7 700 1200 8 800 1100 9 900 1000 10 11 12 13 14 15 16 17 18