Enzymes 1 Flashcards
What is the definition of an enzyme?
Proteins that catalyse specific reactions
Give some functions of enzymes
Digestion Blood clotting Immune system Movement Nerve conduction
How are enzymes categorised?
According to which reaction they promote
Give examples of enzyme categories
Protease
Nuclease
Polymerase
Kinase
Give some examples of enzyme disfunctions that cause disease
Phenylketonuria
Glycogen storage disease
Tay-Sachs disease
Glycosphingolipid
What does phenylketonuria mean?
Cannot convert Phe into Tyr
What does glycogen storage disease mean?
Cannot mobilise (breakdown) glucose
What does Tay-Sachs disease mean?
Defect in processing a membrane ganglioside causing neuronal damage
What is a ganglioside?
A molecule composed of a glycosphingolipid with one or more sialic acids linked on the sugar chain
How does penicillin target enzymes?
Inhibit cell wall synthesis
How does aspirin block enzymes?
Blocks prostaglandin
How does methotrexate block enzymes?
Interferes with the synthesis of DNA precursors
Give the key properties of an enzyme
Increase reaction rate Shows specificity Unchanged at the end of the reaction Do not alter the reaction equilibrium Facilitate reaction by decreasing the free energy of activation of the reaction
How much do enzymes increase the reaction rate by?
10 billion fold
What is the transition state?
The place where if you fall backwards you would have the reactants and if you move forwards you will get the products of the reaction
What is an active site?
It is a 3D cavity that binds substrates with specificity through electrostatic, hydrogen, van def waals and hydrophobic interactions forming an E-S complex
Give evidence for active sites
X-ray crystallography
Kinetic studies of enzyme activity
Give the steps in X-ray crystallography
- make lots of the protein using DNA recombination technology
- get it to crystallise
- put crystals in an intense X-ray beam at a single wavelength
How do you get an image using X-ray crystallography
The X-rays defract into a detector with photographic film on the other side.
You get a diffraction pattern that can b spit through software to get a molecular shape
Why do you need an E-S binding energy?
- to bring molecules together in the active site
- to constrain substrate movement
- to distort substrate so bonds are strained, making it easier to break
- To exclude water From the reaction site, making the reaction go faster
- to provide a reaction pathway of lower energy
What happens when an enzyme is saturated?
The reaction velocity begins to plateau
What does the Michaelis constant measure?
Substrate binding affinity ( how sticky the active site is to the substrate)
How do you get Vmax and Km using a double reciprocal plot?
Measure v at various substrate concentrations
Plot 1/v versus 1/[s]
Intercept on the y axis = 1/vmax
Intercept on the x axis= km
How do competitive inhibitors work?
Compete with the substrate for binding to the active site, forming an EI complex
What is an EI complex?
Enzyme- inhibitor complex
What happens to Km and vmax in the presence of a competitive inhibitor?
Takes more substrate to achieve Vmax/2 but Vmax is the same as the inhibitor can be outcompeted at high concentrations
What is non- competitive inhibition?
Inhibitor binds at a different site so doesn’t compete for binding at the active site
What happens to km and vmax in the presence of a non- competitive inhibitor?
Km is unaltered but vmax is reduced
How does control of gene expression regulate enzymes?
Limits the amount of enzyme made
How does compartmentation regulate enzymes?
Sequences in enzyme polypeptide chain target enzyme to different organelles
How does allosteric regulation regulate enzymes?
Changes the active site to increase or decrease enzyme activity - controls the flux of material through metabolic pathways
How does covalent modification of the enzyme regulate enzymes?
Change enzyme shape and activity by (for example) phosphorylation
What are the properties of an allosteric enzyme?
Multisubunit complexes
Regulatory and catalytic sites on different subunits
Regulation occurs by conformational changes
Exhibits non- Michaelis-menten kinetics
Involved in feedback regulation of metabolic pathways
What shaped curve does an allosteric enzyme give?
Sigmoid
What does novobiocin do?
It’s a competitive inhibitor to ATP as its binding site overlaps the ATP binding site
What do fluoroquinolones do?
Inhibit DNA resealing by gyrase/topo IV - breaks the double strand (reversibly)
