Many proteins can aggregate into amyloid fibrils. Fibril elongation has many sim

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.

a) Describe how kinetics could be experimentally measured.

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

c) Describe the factors 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.

Explanation / Answer

Please find the answers below:

Part a) Enzyme kinetics or aggregation kinetics can be spectrophotometrically analysed. Spectrophotometry is a routinely used laboratory technique which exploits the fact that every biomoleucle has a fixed range of absorbance of light in different physical forms i.e. in solution, as un-aggregated molecules and in aggregates as well. For analysis of kinetics of an aggregation expriment, following steps could be made:

Thus, this spectrophotometric analysis can be easily used to determine the state of solute/protein in a sample.

Part b) Amyloid fibres accumulate to form large aggregates. The rate of amyloid protein accumulation curve can be superimposed to a standard Michelis enzyme kinetic reaction. This degree of superimposition and extent of match between these two states deciphers that amyloid aggregation indeed follows Michaelis-Menten enzyme kinetics. The shape and nature of curve can be further exploited to determine qualitative and quantitative information from this kinetic curve. Here, the substrate will be amyloid protein and product will be amyloid fibres and no catalysis will be performed by an enzyme and only self-aggregation will take place.

Part c) Factors that would affect the conversion of monomer into a fibril will be comparable to a standard Michaelis-Menten enzyme kinetic reaction i.e. :

1. The concentration of the substrate: The concentration of the substrate or the amyloid monomer will determine the extent of amyoloid aggregated. Lesser is the monomer concentration, lesser will be the aggregates made.

2. The feedback inhibition: Any negative feedback inhibition from the amyloid fibrils formed might interfere with the reaction and stop further formation of plaques/fibres. However, positive feedback will promote further formation of fibrils.

Part d) From Michaelis-Menten enzyme kinetic curve, the nature of inhibitor can be identified easily by qualitative assessment. When no inhibitor is used for a reaction, the curve follows a normal enzyme kinetics curve formation. When a competetive inhibitor is used, the curve is shifted slightly downwards in a flat position. The Km remains same here but the Vmax changes. On the other hand, when a non-competetive inhibitor is use, the curve is shifted highly downwards with maintenance of curvature. The Vmax remains same here but the Km changes.

Part e) Sonication is a technique by which proteins are broken down into their basic structures such a secondary or primary sequences. However, sonication also involves slightly heating effect which can lead to aggregation of proteins. Practically, the process of sonication needs to be standardized for every protein being used. However, since amyloid is a fibrous protein by nature, it is difficult to dissipate it into its native sequence by sonication hence the protein aggregates to form fibrils.

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