Question

Many proteins can aggregate into amyloid fibrils. Fibril elongation has many similarities to an enzymatic reaction. Experimental data on insulin amyloid-like fibril elongation suggest that the formation of fibrils follows Michaelis-Menten kinetics as shown in the below figure.

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A.    Describe how kinetics could be experimentally measured.

B.    Describe how amyloid fibers follow the Michaelis-Menten enzyme kinetics.

C.    Describe the factors that would affect the conversion of the monomer into the fibril.

D.    You identify a potential inhibitor of the conversion. Describe how to identify the type of inhibition.

E.    Sonication is commonly used to create amyloid fibril. Suggest a hypothesis as to how sonication facilitates conversion.

Answer 1

Please find the answers below:

Part A: Kinetics of this experiment can be measured experimentally by spectroscopic analysis. The absorbance/fluorescence of amyloid protein is different in monomer form than aggregate form. The relative change in absorbance/fluorescence observed with time and with respect to the monomers left in the solute will give the exact kinetic profile of aggregation of amyloid proteins.

Part B: According to Michelis-Menten enzyme kinetics, the rate of product formation and the rate of substrate depletion are dependent upon the activity of enzyme and the extent of product formation is dependent upon the extent of substrate present. When plotted on graph, it gives a characteristic differential appearance. The rate of amyloid aggregate formation with respect to time when plotted on graph has a similar appearance. This states that amyloid aggregation follows Michelis-Menten enzyme kinetics.

Part C: Similar to Michelis-Menten enzyme kinetics, the rate of reaction would be dependent solely upon the temperature of the system. However, extent of product formation will be dependent upon:

a. The concentration of substrates depletion

b. The concentration of product accumulation

c. Positive/negative feedback loop inhibition

Part D: There can be two types of inihibitors for a reaction, competetive or non-competetive. Graphically, a competetive inihibitor gives an appearance of slightly flat curve just below the curve of normal kinetics whereas the curve for non-competetive inhibition is located much lower than both of them and is sigmoid in nature. Technically, a kinetic inhibition in which the maximum reaction velocity remains unchanged and Micheli's constant changes represents a competetive inhibition. On the other hand, a kinetic inhibition in which the Micheli's constant remains unchanged and the maximum reaction velocity changes represents non-competetive inihibition.

Part E: Sonication is a technique used to degrade proteins in a sample. However, the heating effect of sonication also leads to aggreagation of proteins due to breakdown of primary structure of proteins. In the present situation, sonication might be leading to complete denaturation of the primary polypeptide sequence of the protein thus leading to increase in aggregation of the amino acids. This clumping finally promotes aggregate formation of amyloid proteins.