ANSWER THE FOLLOWING PROBLEM PLEASE (biochem) You have just discovered a new pro

Question

ANSWER THE FOLLOWING PROBLEM PLEASE (biochem)

You have just discovered a new protease in a bacterium you are studying! Based on sequence analysis, you think the following amino acids may be involved in the mechanism: Cys61, His87, Asp115, and Asp134. Based on these potentially important amino acids, you conclude that you have either a cysteine protease (analogous to a serine protease, except that a weaker base can be used to deprotonate the Cys), an aspartyl protease (uses two aspartates for acid/base catalysis), or a metalloprotease (uses Zn2-). 2. Propose a mechanism for the cleavage of a peptide bond by each type of protease. Note that no one mechanism requires all four amino acids. a. Make a table like the one below to summarize the role of each residue in each mechanism, i.e, base, acid, stabilizer, nucleophile, metal ligand, or not involved. Cys Protease Asp Protease Zn2 Protease b. Cys 61 His 87 Asp115 Asp134 You treat the enzyme with a high concentration of EDTA, a metal chelator, and you observe no decrease in function. Which mechanism can you rule out? c. d. Propose a site-directed mutagenesis experiment that will differentiate between the remaining two mechanisms. Based on your experiments, you conclude that your protease is an aspartyl protease. To support your hypothesis, you test the catalytic activity at various pH values. The enzyme is maximally active at pH 6.5, and there is a major decrease in activity at pH 8. Explain this observation. e.

Explanation / Answer

Answer:

a)

Zn2+

Protease

b)

C) metal chelator EDTA finds with free metal ion but here Zn metal ion is bring into active site where it coordinated with 2 his & one glu so this mechanism could be rule out because it doesn't alter the enzyme activity.

d) site-directed mutagenesis is a molecular method use to make intentional change to gene or gene product.

here for cys protease we can create a point mutaion for change cys residue to other amino acid thereby only asp protease do their job but cys protease fails to cleave the peptide bond at Cys 61.

same as above we can do point mutation for asp residue to other amino acid & after do that asp protease is unable to cleave the peptide at specific Asp position at 115 & 134.

protease Mechanism Cys protease It catalyse the hydrolysis of peptide bond by deprotonation of a thiol in the active site of enzyme by an adjacent amino acid having basic side chain usually Histidine. Asp protease here two aspartate residue disposed to the opposite site of the peptide bond. one asp residue act as a base for H-OH bond and do nucleophillic acttack on water , on the other hand second Asp residue act as a acid to protonate the departing amine product , thus,cleave the bond.

Zn2+

Protease

One Zn atom is coordinated specifically to two His residue and one glu side chain that bring super matal divalent cation into the active site where H-OH bound with Zn & dissociate in the form of Zn-OH ready to attack the substrate peptide bond.

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