Enzymes background and Proteases for Hydrolysis

pharm 2001

Catalyst

a substances that speeds up a chemical reaction without being altered (changed) / consumed during a reaction

enzyme

a protein that acts like a biological catalyst

What can catalytic efficiency of an enzyme do to biochemical reactions, and what does it do ?

• increase the speed reaction of biochemical reactions

• efficiency of biochemical reactions

• This causes for many reactions to occur in picoseconds

Enzyme reaction conditions are ___ (something that lacks the strength to react chemically). This is because chemical reactions have different properties such as:

Enzyme reactions are inert

• mild temp, pH, and aqueous environments

Enzymes are ____, meaning they won’t produce any products or will not undergo any reaction with__

• They are specific, undesirable functional groups or substrates

• Think about substrates that need to be the same shape for the enzyme to be activitated

Reversible enzyme inhibitors

non-covalent bonds with the enzyme and inhibitor

What type of interactions/bonds are examples of reversible enzyme inhibitors

• hydrogen bonds

• hydrophobic interactions

• electrostatic interaction

what are examples of inhibitors that are considered reversible enzyme inhibitors

competitive, uncompetitive, and non-competitive

Competitive inhibition

• substrate and inhibitor cannot bind at the same time ( compete for the active site)

non-competitive inhibition

• do not compete for the active site

• inhibitor binds to the allosteric site

uncompetitive inhibitor

• The inhibitor binds to the enzyme-substrate complex (meaning an enzyme that needs two substrates to produce a product), where one substrate is already attached to the enzyme

• blocks it from producing a product

How to overcome competitive inhibitors

• higher concentration of substrate than inhibitor

What does an inhibitor structure resemble

• substrate/transition state structure

substrate and cofactor mimics resemble what

• natural substrate or cofactors of targeted enzymes

what do the substrate and cofactors mimics do and what does it prevent

• binds with the active site of the enzymes

• prevents from natural substrate/cofactors from interacting in the active site

• so they are known as competivite inhibitors

what are substrate and cofactor mimics used as ___

• drugs or probes for in enzymes

Caveat means

selectivity

Transition state

the most unstable structure that has the highest energy state along the reaction path from substrate to product

Enzymes can bind to substrates and __

transition state

Why can enzymes bind to transition state?

• enzymes are optimized and evolved to coordinate to bind to them

Transition state mimics are what type of inhibitors

competitive

transition state mimics can make great _____ and ______

• drugs

• chemical probes

Orthosteric site

the primary binding site on an enzyme or receptor that ligand interacts with

Allosteric site

another potential binding site that isn’t the orthosteric site

Traditionally probes/drugs are developed to target and bing ____ site

orthosteric site

Why did probes/drug traditional target and bind to the orthosteric site

easier to identify orthosteric sites and target orthosteric site but it was less selective

why is there an increase in attempting to identify and target allosteric site

• more selective for a specific receptor or enzyme subtype/isoform

• less likely to develop mutations

Reversible inhibitors ( dont get this confused with reversible enzyme inhibitors, tho they are closely related)

• compounds (think about drugs or probes) that bind temporarily through noncovalent interactions with the binding site

• so drugs and probes are reversible

Residence time

how long reversible drug stays bound to its target

How are drugs/probe that are reversible inhibitors measure potency (the concentration of drug aka dose needed for the activity of the drug to have an effect)

• affinity and residence time

Irreversible inhibitors

• drug that form covalent bonds with the binding site on their target

Examples of irreversible inhibitors

• covalent inhibitors

• suicide inhibitors

• Penicillin antibiotics

Penicillin antibiotics are ____ and do they do

• beta lactams

• inhibit cell wall biosynthesis

what is penicillin easily deactivated by?

• bacterial beta-lactamase enzymes

What do you need to add to penicillin to make an irreversible inhibitor?

Clavulanic acid

what are known as substrate mimic

• penicillin and clavulanic acid

Protease

cleaves (split apart) peptide bonds (amide bonds) through hydrolysis

• keep in mind that they are also enzymes

How protease enzymes cleave the bonds?

• they use side chains in the active site use the water to break them apart (remember they use break the bonds through hydrolysis)

what enzymes are examples of protease

• Peptidase, proteinase, proteolytic enzymes

what roles do proteases play across all organisms

• essential for biological signaling, protein degradation/recycling, cellular development, disease onset and progression

How are proteases targetable enzymes exploited

• with small molecules as chemical probes or drugs

In proteases the ___ bond and ___ geometry are resistant to chemical hydrolysis

• planar amide bonds

How can protease break apart amide bonds?

Overcome the stabilizing feature of the structure (planar shape)

what shape does the proteases need to change into to break the amide bond and loos planarity?

• tetrahedral

How are proteases evolved to stabilize what

• non-planar and high-energy transition

what do tetrahedral transition state mimic do

• bind tightly and make great inhibitors

HIV Protease function

• essential for processing HIV precursor protein (inactive proteins) to active forms for viral replication

what is the structure of the HIV protease it has two identical subunits of ____

• olbigate homodimer of two identical subunits

where is the active site located between ___ in the HIV Protease? Describe its symmetry

two subunits and is symmetrical

How does the HIV Protease use to activate water as the nucleophile?

Uses two aspartate residues from each subunit

not catalytic triad

How is the selectivity of HIV PRotease or viral protease helps with drug targets as inbhitiors to target the viral enzyme

• viral protease structures are different from human proteases

• which is why they are good to develop drugs targets as ihibitors because they wont afffect human proteases

What is the mechanism HIV protease uses for selectivity

• break apart between Phe and Pro