Borders able below shows some of the amino acids from the human, rat, and fruit

Question

Borders able below shows some of the amino acids from the human, rat, and fruit fly enzymes. In addition, mutants (indicated by the asterisks) have been engineered (via site-directed mutagenesis) for each enzyme. The unfolding Tm, the Ks (binding constant, k1/k-1) for phosphate, and the Hill coefficient for allosteric regulation of catalysis (k2) are given; the R and T states bind substrate equivalently but the T state catalyzes the subsequent reaction much more slowly than the R state. The concentration of Pi in each organism is 1 mM. Mutant sequences and their effects are in bold in the table Enzyme Human Human* Rat Rat* Fly Amino Acid Sequences: Residue # (where it is) 27 (core3 (core 57 (surface) 72 (surface) Gly Val Gly Ala Ala lle lle Ala Val Phe Phe Phe Phe Tyr Enzyme Properties Tm (C) K(Pi), mM Hill n 45 32 45 45 45 20 0.5 0.5 0.5 10 0.5 0.5 2.7 2.7 2.7 2.7 2.7 ys ys ys Ala Ar rg ys Ar rg (a) The crystal structure of the Human* mutant enzyme shows that a cavity has been created in the hydrophobic core by changing Val Ala. Why might this destabilize the folding of the protein? (b) In the Rat* enzyme, switching lle and Ala has no effect on stability. Why not (suggest a plausible reason)? (c) If switching the amino acids at residues 27 and 30 has no effect on stability, and if it were shown furthermore to have no effect on function, why hasn't this mutation occurred naturally? (d) Clearly the side chains of residues 27 and 30 interact. If they are part of a repeating secondary structure (not a turn) in the protein core, is it more likely to be an a-helix or a b-sheet? Why? (e) Explain why the Val Lys mutation in Fly" is strongly destabilizing

Explanation / Answer

The cavity would form because of the nonconservative substitution. The protein structure of human enzyme cannot bear the substitution of Valine to alanine. That is the case of Conservative substitution. The protein structure of rat enzyme can bear the interchange of Isoleucine to alanine. However, the conservative substitution can only possible when substitution occurs within the same property group of amino acid. Yes, if substitution has no effect and enzyme functionally same as the original one, then the mutations occur naturally and are example of nonconservative substitution. Yes, the secondary structures are two types: alpha chain and beta sheet. Therefore, if 27 and 30 residues interact in a secondary structure, they would participate in either alpha chain or beta sheet. Valine is a hydrophobic amino acid while Lysine is a positive charged amino acid. Therefore, substitution in such case would cause greater destabilization.

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