Question

The enzyme that synthesizes the neuropeptide discussed in question 1 is tyrosyl-tRNA synthetase. (PLEASE ANSWER ALL 3 PARTS!!!!!!!!)

A) This enzyme requires ATP to synthesize the dipeptide, but in the ribosome peptide bond synthesis requires no ATP, why?

B) You are a scientist who wants to study this enzyme-outline a set of experiments you would perform to produce recombinant protein.  You must be specific in your strategy. (i.e. you can’t just say that you will purify the protein-you must indicate how).

C) tyrosyl-tRNA synthetase makes a tyrosyl adenylate intermediate (essential an AMP-conjugated to the amino acid). Within the enzyme a Cys 35 hydrogen bonds to the 3’OH in AMP. Draw this hydrogen bond interaction and indicate the impact of mutating the Cys to Ala for this interaction.

Answer 1

A) tyrosyl-tRNA synthetase requires ATP for synthesising the dipeptide as it needs to first activate the substrate i.e. tyrosine by reaction with ATP to form high-energy intermediate tyrosine adenylate, with the release of pyrophosphate.

While ribosome peptide-bond synthesis doesn't require ATP because the free energy of hydrolysis of PPi generated in the first step, is highly negative to drive the reaction spontaneously.

B) Experiments to produce a recombinant protein:

• Incorporation of the 'gene of interest' for protein in the expression vector by cloning.
• Generation of the competant cells and transformation using electroporation.
• Selection of the positively transformed clones using antibiotic exclusion assays or blue-white screening.
• Screening of the individual colonies using Gel electrophoresis based assays.
• Expansion of the positive clones using sub-cloning experiments.
• Protein produced are analyzed after cell lysis experiments.
• Purification of the recombinant protein is done using the appropriate chromatography, based on the characteristics of the transformed protein.

C) Cystein present in the enzyme tyrosyl-tRNA synthetase forms a hydrogen bond between -HS group and the H of the OH- group present in AMP. So, when Cystein is replaced by alanine, the thiol group also get replaced and hence, no H-bonding remains between AMP and the enzyme.