Enzyme Reaction & Mechanism (Module 1 Lecture 5)

Question 1

What are Four Classes of Proteases?

Answer 1

1. Serine Proteases
2. Cysteine Proteases
3. Aspartyl Proteases
4. Metalloproteases

Question 2

What are Some Examples of Serine Protease?

Answer 2

1. Trypsin
2. Chymotrypsin
3. Elastase

Question 3

Contrast Serine Proteases’ Substrate Specificity .

Answer 3

1. Trypsin uses a COO- to bind to arginine and lysine residues (which are positively charged).
2. Elastase uses bulky Valine side groups so that only alanine, serine, and glycine residues are small enough to bind to the active site.

Question 4

What Do ALL Serine Proteases Have in Common?

Answer 4

A triad with Serine residue as the active NUCLEOPHILE

Question 5

What is a Major Role of Cysteine Proteases?

Answer 5

Cysteine-Aspartic Proteases (Caspases) play a huge role in apoptosis (cell suicide!)

Question 6

What are TWO Major Roles of Metalloproteases?

Answer 6

Tissue remodeling (good) and cancer metastasis (bad) involve Matrix Metalloproteases (which use zinc and break peptide bonds of non-terminal amino acids (i.e. endopeptidases)).

Question 7

Explain Chymotrypsin’s Catalytic Mechanism.

Answer 7

Asp-102’s COO- hydrogen bonds with Hystidine-57’s secondary amino group and the other N in the hystidine ring attacks Serine-195’s H in it’s alcohol, stealing it and rendering the O- a nucleophile, which is ready to attack the CARBONYL group on a substrate.

Question 8

What are the Three Amino Acids in Serine Proteases’ “Catalytic Triad?”

Answer 8

1. Aspartine-102
2. Histidine-57
3. Serine-195

Question 9

Explain Chymotrypsin’s Substrate Specificity.

Answer 9

Chymotrypsin uses hydrophobic amino acid side chains next to Ser-195 so the only substrates that will attach will have aromatic or large hydrophobic side chains (with amino acids like tyrosine, tryptophan, phenylalanine, and methionine).

Question 10

What is a Function of Trypsin?

Answer 10

Trypsin is often used in cell cultures and is a digestive enzyme that catalyzes the hydrolysis of lysine and arginine resides ONLY after their carboxyl sides and before the subsequent amino acid’s amino side.

Question 11

What is a Function of Thrombin?

Answer 11

Thrombin is an enzyme that helps blood clot by hydrolyzing (cleaving) between the carboxyl side of an arginine and the amino side of a glycine when they appear in specific sequences (Arg-Gly).

Question 12

How Many Steps Does Chymotrypsin Catalysis Use? Explain.

Answer 12

Two steps:

1. After the nucleophilic attack, the protein is cleaved and the COO- is covalently bound to Serine as a covalent intermediate (acyl-enzyme complex). The NH2 is released.
2. Then de-acylation occurs when the Ser-O-C=O is flushed with water and released from the serene (Serine takes the H back on his O- and the C=O also gets an OH and becomes a carboxylic acid again).

Question 13

Define Protease.

Answer 13

An enzyme that cleaves proteins (AKA proteolytic enzymes).

Question 14

Define Proteolytic Enzymes.

Answer 14

Enzymes that cleave proteins via hydrolysis (AKA proteases).

Question 15

Define Dehydrogenases.

Answer 15

Enzymes that remove hydrogens from substrates.

Question 16

Explain the Role of Lactate Dehydrogenase.

Answer 16

Lactate dehydrogenase is an enzyme that removes two hydrogen atoms from lactate (C3H6O3) and passes them to NAD+ during glycolysis, creating pyruvate (C3H4O3) and NADH and a proton.

Question 17

What is NAD+?

Answer 17

Nicotinamide Adenine Dinucleotide - an electron transport molecule that serves as a cofactor with the vitamin niacin in it.

Question 18

Examples of Measuring Enzyme Activity Using Changes in Absorption of Light:

Answer 18

1. NAD+ –> NADH happens in tandem with a change in optical density.
2. Dehydrogenase activity rates can be measured at the optical density, or absorbance, at a wavelength of 340nm (NADH is more optically DENSE than NAD+).