Enzyme Kinetics

Chemical reaction calalysed by malate dehydrogenase

NAD+/NADH

• cofactor

• helps enzyme enlarge thier fucntionality beyond that of the natural amino acid side chain residues

• It is the redoc currecny

In which biological molecules do you find phosphate groups?

• DNA

• RNA

Where is Micotinamide found

• functional group of NAD+

Role of NAD+/NADH

• NAD⁺ is used for the oxidation of a substrate.

• NADH is used for the reduction of a substrate.

What wavelgnth is used for NADH

• One that is the greatest difference to NAD+

• so that we can distinguish the change

Continuous assay

• assay gives an uninterupted reading of substrate or procuct

Discontinuous assay

• samples are taken, the reaction stoppped and then the concentration of substrates/products determined

What could be the potential reasons why the absorbance increase over time is not entirely linear?

1. The reaction product produced in the reaction is structurally similar to the starting material and thus a fraction of the product does not leave the active sites. The product residing in the active site then blocks the entry of a new substrate molecule.

   • Product inhibition→ systematic deviation

2. The enzyme activity decreases after a number of turnovers because the enzyme gets less active.

   • Denaturation, partial unfolding

HOw make a michalis menem plot

V0 against suubstrate concentration

• The Km is the substrate concentration when vmax/2

Other way→ Lineweaver burk plot

• LInerlised form

1/v0 against 1/substrate concentration

Where is the Km read?

• -1/Km is the x intercept

What is Kcat

Vamx/[E]

Which enzyme (MDH1 or MDH2) shifts the equilibrium constant, K_eq, more in the direction of product?

• The correct answer is: The equilibrium constant is the same for both enzymes

• The equilibrium constant is dependent on the thermodynamics of the reaction; enzymes change the kinetics by lowering the transition state energy, but cannot change the equilibrium thermodynamics.

MDH is an important enzyme, with several roles in metabolism - it is found in a number of different locations (in chloroplasts and in mitochondria as well as in the cytoplasm). Using the following data, calculate the number of molecules of malate and of malate dehydrogenase in the cytoplasm plus organelles of a single leaf cell.

• 1g leaf tissue has 2 x 10⁶ cells and 2 mg protein

• 1 leaf cell has a volume of 6 x 10^-5 μl of which 10% is cytoplasm plus organelles

• The concentration of malate in the cytoplasm plus organelles is 1mM

• Malate dehydrogenase has a M_r of 70,000 and is 0.1% of the total protein in the cell.

• Avogadros number is 6 x 10²³.

• I can do this woo