chapter 13 section 8

Question 1

Why is designing enzymes desirable?

Answer 1

To create catalysts for industry, medicine, or environmental cleanup.

Question 2

What is one common approach to enzyme design?

Answer 2

Mutate active-site residues in a known enzyme (in vitro mutagenesis).

Question 3

What is a fully new enzyme design approach?

Answer 3

Build a protein from scratch using computer modeling (in silico).

Question 4

What is the main challenge of in silico enzyme design?

Answer 4

Ensuring correct folding, stability, and activity in cells

Question 5

What is semirational enzyme design?

Answer 5

Combine rational active-site mutation with directed evolution in bacteria.

Question 6

What software is popular for in silico enzyme design?

Answer 6

Rosetta protein modeling suite.

Question 7

What catalytic efficiency has been achieved by designed enzymes?

Answer 7

kcat​/Km​>10^5M−1s−1

Question 8

What was the effect of these mutations on enzyme activity

Answer 8

The mutant efficiently converted cis-dichloroepoxyethane to Cl⁻ ions and glyoxal, greatly increasing 𝑉max/Km

Question 8

How was a designer enzyme created for degrading chlorinated epoxides?

Answer 8

By site-directed mutation of bacterial epoxide hydrolase at three active-site residues (F108, I219, C248) to produce an F108L/I219L/C248I mutant.

Question 9

Why is this designer enzyme important?

Answer 9

It helps degrade toxic chlorinated epoxides, aiding in water bioremediation.

Question 10

What reaction is used as a model for designer enzymes?

Answer 10

The Kemp elimination reaction.

Question 11

What protein scaffold is commonly used for Kemp eliminases?

Answer 11

TIM α/β-barrel structures.

Question 12

How are active sites optimized in Kemp eliminases?

Answer 12

By testing bases (Asp, Glu) and forming His-Asp/Glu dyads.

Question 13

What methods improve Kemp eliminase activity?

Answer 13

Computational design plus directed evolution.

Question 14

How much has catalytic activity improved in KE designer enzymes?

Answer 14

over 700-fold compared to earlier versions.